WO2014171767A1 - Procédé pour distinguer l'authenticité du miel à l'aide de la détection d'adn - Google Patents

Procédé pour distinguer l'authenticité du miel à l'aide de la détection d'adn Download PDF

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Publication number
WO2014171767A1
WO2014171767A1 PCT/KR2014/003369 KR2014003369W WO2014171767A1 WO 2014171767 A1 WO2014171767 A1 WO 2014171767A1 KR 2014003369 W KR2014003369 W KR 2014003369W WO 2014171767 A1 WO2014171767 A1 WO 2014171767A1
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honey
dna
sample
bee
plant
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PCT/KR2014/003369
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English (en)
Korean (ko)
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김동호
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주식회사 제놀루션
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Publication of WO2014171767A1 publication Critical patent/WO2014171767A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method of detecting plant-derived DNA and honeybee-derived DNA from honey to determine a sample containing no pure honey and heat-treated honey, specification honey, diluted honey, and pure honey at all.
  • Honey is honeycomb honey that honeybees collect natural products such as flower honey and sap and store them in honeycomb, and honey is honey that is not added to other foods or food additives such as pollen, royal jelly, sugar, sweetener.
  • Honeycomb honey means that honey bees collect natural materials such as flower honey and sap and store them in the honeycomb and then seal all or part of the honeycomb.
  • the honeycomb is a honey bee that collects natural honey such as flower honey and sap. It refers to matured things that have been stored in honeycomb (Food Code).
  • Honey contains sugars such as glucose and fructose as well as ideal nutrients such as vitamins, proteins, minerals and amino acids that are unique to pollen, preventing and treating fatigue, anemia, diabetes and liver disease, relieving hangovers and improving beauty.
  • fructose which is a main ingredient in honey, is consumed in the food industry as a sweetener because it has a fast absorption rate and high sweetening effect compared to sucrose.
  • honey is honey that is produced by bees eating sugar and storing it in the process of raising bees.
  • Sugar like nectar, is collected by bees and then transferred from bees to bees, digested by enzymes in the body, stored in honeycombs, dehydrated and dried.
  • honey has a shorter stay in the body than honey and lacks various nutrients derived from honey, honey's quality is much lower than honey.
  • Another example of fake honey is honey diluted with impurities such as rice syrup or corn syrup. Rice syrup and corn syrup are much cheaper than pure honey, but, like pure honey, they contain fructose and have a similar sweetness.
  • honey added to these rice syrup or corn syrup is also significantly lower than various honey content.
  • honey autonomous labeling allows labeling of 13C / 12C isotope ratio autonomously. Plants are classified as C3, C4, CAM plants according to the method of fixing CO 2 in photosynthesis. Plants such as rice, wheat, and corn are C3 plants, and the flowering plants that are the source of wheat are mainly C4 plants.
  • honey honey diluted with C3 plant rice or corn syrup and sugar honey produced with sugar water has a relatively high 13C / 12C isotope ratio.
  • acacia honey, wildflower honey, rapeseed honey and chestnut honey have a carbon isotope ratio of -23.5 or less, miscellaneous honey -22 or less, honey with added rice syrup, -9 to -11 or less, and honey honey -12 It is known to appear as follows.
  • the authenticity of honey using the 13C / 12C isotope ratio requires complex equipment and skilled manpower.
  • the syrups of C4 plants such as beet are added, the false 13C / 12C isotope ratio is low even though it is fake honey. There is a limit.
  • honey that has undergone excessive heat treatment is also a problem as the quality is poor as a variety of fake honey.
  • Mature honey has a very high osmotic pressure, so bacteria and fungi do not grow and can be stored for a long time, but immature honey that has been pulverized before being sufficiently dehydrated is easily deteriorated.
  • beekeepers are concentrating on selling prematurely mature immature honey to high-temperature concentrators and drying them due to delivery schedules.
  • Honey treated with a high-temperature concentrator has significantly lower nutrient content than natural honey, which is fully matured after nutrients are destroyed during heat treatment.
  • An object of the present invention is to provide a method for discriminating pure honey and a sample containing no heat treated honey, specification honey, diluted honey, and pure honey by detecting plant DNA and bee DNA in honey.
  • the present invention provides a method for determining the authenticity of the honey sample by detecting plant DNA and bee DNA in the honey sample.
  • determination of the authenticity of the honey sample means to determine the sample that does not contain pure honey and heat-treated honey, specification honey, diluted honey, and pure honey at all.
  • Honey contains a large amount of plant DNA from pollen origin.
  • using a conventional extraction method requires a lot of time and effort for each sample, it is difficult to use for various sample analysis. It also assumes that honey will contain somatic cells of bee origin, as all honey will be fed back from the semi-digested state after honey bees have received nectar of plant origin.
  • Applicants' extraction method among the various extraction methods can simultaneously extract DNA of plant pollen origin and bee somatic origin contained in the sample.
  • 'pure honey' means honey prepared purely from flower honey by bees.
  • 'heat-treated honey' refers to honey that is artificially heat-treated in the manufacturing process in order to replace the enrichment effect through the ripening process.
  • the 'specific honey' means an artificially produced honey that lacks the content of flower honey originated from a flower by providing a bee with a sugar-containing diet.
  • 'diluted honey' means honey prepared by mixing and adding a substance containing sugar such as some rice syrup and corn syrup to pure honey.
  • sample which does not contain pure honey at all refers to a sample made of only a substance containing sugar such as rice syrup and corn syrup without containing pure honey at all.
  • the high and low degree of plant DNA or bee DNA content is determined based on pure honey as the reference honey.
  • the detection of plant DNA and bee DNA can be performed using quantitative PCR.
  • the present invention is a.
  • the step 1) may be more preferably performed using Geno-Honey DNA Kit and magnetic beads of Genoa Korea.
  • Other DNA separation methods that can be used include Qiaquick PCR Clean up Kit (Qiagen) or DNeasy Tissue Kit (Qiagen).
  • Qiaquick PCR Clean up Kit Qiagen
  • DNeasy Tissue Kit Qiagen
  • the method of separating DNA is not limited thereto.
  • the process of detecting plant DNA and bee DNA in step 2) may be performed in any order or simultaneously.
  • the detection of step 2) may be performed using quantitative PCR.
  • the plant common primer pair of SEQ ID NO: 1 and 2 and the bee common primer pair of SEQ ID NO: 3 and 4 can be used.
  • primers that can be used are not limited to this, and may be used in the future to replace additional common primers for plants and bees. In this case, the same sample can be used to replace the difference between the value and the current primer.
  • honey may be determined to be honeycomb honey.
  • Sugar contains trace amounts of plant DNA, and sugar-supplied honeybees visit flowers very low.
  • the honey may be determined to be honey that has been heat-treated at a temperature of about 80 ° C. or more and less than 90 ° C.
  • Plant-derived DNA because of its pollen origin, is wrapped in a thick cell wall, making it more heat resistant than honey bee DNA. This is because the plant DNA content is significantly maintained while the bee DNA content is significantly reduced when heat treated at a temperature of about 80 ° C. or more and less than 90 ° C.
  • the honey may be determined to be honey that has been heat-treated at a temperature of about 80 ° C. or more and less than 90 ° C.
  • Plant-derived DNA because of its pollen origin, is wrapped in a thick cell wall, making it more heat resistant than honey bee DNA. This is because the plant DNA content is significantly maintained while the bee DNA content is significantly reduced when heat treated at a temperature of about 80 ° C. or more and less than 90 ° C. This also includes the case where pure honey is diluted with some impurities.
  • the honey sample in step 1) is 1 to 3 ml
  • the separated DNA in step 2) is 5 to 10 ⁇ l of the DNA solution isolated from the honey sample and the detection is sequence It can be performed by quantitative PCR using the plant consensus primer pair of No. 1 and SEQ ID NO: 2 and the bee consensus primer pair of SEQ ID NOs: 3 and 4. It is apparent to those skilled in the art that such quantitative PCR conditions may be variously set according to a person skilled in the art, and thus, the Ct value detected may vary. Thus the quantitative PCR conditions are not limited thereby.
  • the honey sample is heat-treated at 90 ° C. or higher, specification honey, diluted honey, or pure honey at all. Provides a method for discriminating between samples that do not contain.
  • the honey sample is heat-treated at 80 ° C. or higher, specification honey, diluted honey, or pure honey at all. Provides a method for discriminating between samples that do not contain.
  • the honey sample is provided with a method of discriminating the sample that contains no honey or pure honey heat-treated at 90 ° C or more.
  • the honey sample is determined as diluted honey. To provide.
  • the Ct value for the plant DNA in the quantitative PCR condition is a good quality pure honey containing a large amount of plant DNA, the smaller the Ct value based on 27, while the higher the Ct value is diluted honey, It can be judged as honey or heat-treated honey at a temperature above 90 ° C.
  • the Ct value for the bee DNA in the quantitative PCR conditions is a good quality honey containing a large amount of bee DNA as the Ct value is smaller, based on 25, while the honey is diluted, the higher the Ct value, or It can be judged that honey is heat-treated at a temperature of 80 ° C or higher.
  • the extraction of DNA from each of the two species contained therein and the measurement thereof can be used to determine the quality of honey according to the relative values.
  • the method of the present invention can process multiple samples simultaneously, does not consume large amounts of samples, and can be performed simply and quickly. Therefore, it is possible to ensure the trust of consumers and encourage the production of pure honey by enabling the effective authenticity of honey.
  • 1 is a diagram showing changes in plant DNA and bee DNA content in honey according to the heat treatment.
  • Figure 2 is a diagram showing the changes in plant DNA and bee DNA content in the honey product marketed when stored for 0, 2, 4, 6 months from the date of manufacture.
  • Acacia honey was harvested from a honeycomb located more than 100 meters from a forest composed of more than 70% acacia trees in Hwasun, Jeollanam-do, Korea. After wax capping in a honeycomb for 35 days, the beeswax sealed with a heated wax knife over July 15 to 20, 2012 was cut out and harvested by centrifuging the honeycomb.
  • the sample names of the honey honey were acacia honey I and II, respectively.
  • honey was harvested.
  • the sugar was dissolved in warm distilled water to prepare an aqueous 30% sugar solution, which was placed in a 30 ⁇ 40 cm Pyrex glass plate located within 10 cm of the honeycomb inlet for 7 days.
  • After waxing in a honeycomb 35 days after sugar water supply (wax capping) was sealed by cutting the beeswax sealed with a beeswax knife heated from 15 to 20 July 2012 and centrifuged the honeycomb.
  • the sample names of the honey honey were set as the specification honey I, II and III, respectively.
  • miscellaneous honey was harvested from a honeycomb located about 1.2 km away from various flowers and plants. After wax capping in a honeycomb for 35 days, the beeswax sealed with a heated wax knife over September 20 to 10, 2012 was cut out and the honeycomb was centrifuged. The sample names of the honey that was sampled were miscellaneous honeys I and II, respectively.
  • the honey that was collected was not exposed to sunlight and stored at room temperature.
  • DNA was extracted from the honey samples prepared in 1-1 using Geno-Honey DNA kit manufactured by Genol.
  • the specific extraction process is as follows.
  • Each honey sample was first transferred to a 15 ml Falcon tube by about 3 ml using a 1 ml pipette or poured.
  • the volume of whole honey was measured after centrifugation at 1,000 rpm for 1 minute with a vertical clinical centrifuge. Two times the volume of the measured volume was added to distilled water warmed to 37 ° C. before mixing to dissolve. If not soluble, stir with a metal rod to dissolve completely. 1.5 ml of this solution was taken and transferred to a microtube, followed by centrifugation at 14,000 rpm for 2 minutes. After discarding the supernatant, 1.5 ml of the same solution was added again and centrifuged again.
  • the results for each sample are shown in Table 2 below.
  • the Ct value is the average value obtained after three independent analyzes. Smaller Ct values indicate higher DNA content.
  • honeybee DNA and plant DNA contents change when commercially available honey products are stored for a long time
  • two samples of honey products produced in Korea K12 samples of miscellaneous honey and K17 samples of acacia honey stored at room temperature. For information, see Table 3 below) for 6 months from the day of production.
  • honeybee DNA and plant DNA were detected using quantitative PCR.
  • the honey bee DNA and plant DNA content in the honey can be a marker for determining the authenticity of the honey even after long-term storage.
  • Bee honey and plant DNA were measured by quantitative PCR as described in Example 1 above by purchasing 23 kinds of domestic honey products at a grocery store or a department store in Seoul, South Korea. Specific data are shown in Table 3 below.
  • the Ct value is the average value obtained after two independent analyzes.

Abstract

La présente invention concerne un procédé pour distinguer l'authenticité d'un échantillon de miel par la détection d'un ADN d'une plante et d'un ADN d'abeille dans l'échantillon du miel. Il est possible de distinguer le miel pur, le miel thermotraité, le miel fait à partir de sirop de sucre, le miel dilué, et un échantillon ne contenant aucun miel pur par l'utilisation du procédé de la présente invention. Une pluralité d'échantillons peuvent être simultanément traités par l'utilisation du procédé de la présente invention et le procédé de la présente invention peut être simplement et rapidement réalisé sans consommer une grande quantité d'échantillons. Par conséquent, il est possible de garantir la confiance d'un consommateur et d'encourager la production de miel pur en permettant l'authenticité du miel d'être efficacement distinguée.
PCT/KR2014/003369 2013-04-17 2014-04-17 Procédé pour distinguer l'authenticité du miel à l'aide de la détection d'adn WO2014171767A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101747243B1 (ko) * 2015-05-22 2017-06-14 대한민국 천연 벌꿀 내 쌀시럽 혼합 여부의 판별방법
US11162950B2 (en) 2018-11-29 2021-11-02 International Business Machines Corporation Zonal nanofluidic anti-tamper device for product authentication
US11164190B2 (en) 2018-11-29 2021-11-02 International Business Machines Corporation Method for product authentication using a microfluidic reader
CN113621731A (zh) * 2021-08-26 2021-11-09 中国农业科学院蜜蜂研究所 一种贵州蓝莓蜜的产地溯源和鉴别方法
CN113699220A (zh) * 2021-08-26 2021-11-26 中国农业科学院蜜蜂研究所 一种以地域植物源鉴别蜂蜜及蜂蜜产地溯源的方法
KR20220022730A (ko) * 2020-08-19 2022-02-28 경기대학교 산학협력단 토종꿀에서 어피니티 컬럼을 이용한 잔류 유전자 분리방법
KR20220026093A (ko) * 2020-08-25 2022-03-04 경기대학교 산학협력단 사양꿀에서 어피니티 컬럼을 이용한 잔류 유전자 분리방법
WO2023093731A1 (fr) * 2021-11-23 2023-06-01 中国农业科学院蜜蜂研究所 Procédé d'identification de degré de maturité de miel d'acacia
US11710005B2 (en) 2018-11-29 2023-07-25 International Business Machines Corporation Use of microfluidic reader device for product authentication

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101747243B1 (ko) * 2015-05-22 2017-06-14 대한민국 천연 벌꿀 내 쌀시럽 혼합 여부의 판별방법
US11162950B2 (en) 2018-11-29 2021-11-02 International Business Machines Corporation Zonal nanofluidic anti-tamper device for product authentication
US11164190B2 (en) 2018-11-29 2021-11-02 International Business Machines Corporation Method for product authentication using a microfluidic reader
US11710005B2 (en) 2018-11-29 2023-07-25 International Business Machines Corporation Use of microfluidic reader device for product authentication
KR20220022730A (ko) * 2020-08-19 2022-02-28 경기대학교 산학협력단 토종꿀에서 어피니티 컬럼을 이용한 잔류 유전자 분리방법
KR102371559B1 (ko) 2020-08-19 2022-03-07 경기대학교 산학협력단 토종꿀에서 어피니티 컬럼을 이용한 잔류 유전자 분리방법
KR20220026093A (ko) * 2020-08-25 2022-03-04 경기대학교 산학협력단 사양꿀에서 어피니티 컬럼을 이용한 잔류 유전자 분리방법
KR102371553B1 (ko) 2020-08-25 2022-03-07 경기대학교 산학협력단 사양꿀에서 어피니티 컬럼을 이용한 잔류 유전자 분리방법
CN113621731A (zh) * 2021-08-26 2021-11-09 中国农业科学院蜜蜂研究所 一种贵州蓝莓蜜的产地溯源和鉴别方法
CN113699220A (zh) * 2021-08-26 2021-11-26 中国农业科学院蜜蜂研究所 一种以地域植物源鉴别蜂蜜及蜂蜜产地溯源的方法
WO2023093731A1 (fr) * 2021-11-23 2023-06-01 中国农业科学院蜜蜂研究所 Procédé d'identification de degré de maturité de miel d'acacia

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