WO2014171767A1 - Method for determining authenticity of honey using dna detection - Google Patents

Abstract

The present invention relates to a method for determining the authenticity of a honey sample by detecting a plant DNA and a honeybee DNA in the honey sample. It is possible to distinguish pure honey, heat-treated honey, honey made from sugar syrup, diluted honey, and a sample containing no pure honey by using the method of the present invention. A plurality of samples can be simultaneously treated by using the method of the present invention, and the method of the present invention can be simply and rapidly carried out without consuming a large quantity of samples. Therefore, it is possible to guarantee consumer confidence and encourage the production of pure honey by enabling the authenticity of honey to be effectively distinguished.

Description

Authenticity Determination of Honey Using DNA Detection

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. It is known to show excellent efficacy in promoting children's development and the like, and 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.

However, due to the lack of wheat and the high production cost of pure honey, various fake honeys are on the market. A representative example of fake honey is sugar-fed honey. 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. However, because sugar 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. However, honey added to these rice syrup or corn syrup is also significantly lower than various honey content.

When the market price is less than 1/5 of the pure honey, the fake honey is sold as pure honey, which is frequently sold. As a result, consumers' confidence is lowered and the beekeeping industry is threatened. Korea Food and Drug Administration introduced honey autonomous labeling system from 2009.08.01. Operate. 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. Pure honey has a low 13C / 12C isotope ratio, and 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. For example, 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. However, the authenticity of honey using the 13C / 12C isotope ratio requires complex equipment and skilled manpower. When 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.

In addition, as a method of determining the authenticity of honey, multivariate analysis of chemical substances contained in honey and counting and pollen counting of pollen in honey have been reported.However, it takes a great deal of time and effort to process a large amount of samples. It cannot be used realistically.

In addition, the 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. As a result, 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.

In order to achieve the above object, 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.

In the present invention, "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. However, 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.

In the present invention, 'pure honey' means honey prepared purely from flower honey by bees.

In the present invention, '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.

In the present invention, 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.

In the present invention, 'diluted honey' means honey prepared by mixing and adding a substance containing sugar such as some rice syrup and corn syrup to pure honey.

In the present invention, the "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.

In the present invention, the high and low degree of plant DNA or bee DNA content is determined based on pure honey as the reference honey.

In the method of the present invention, the detection of plant DNA and bee DNA can be performed using quantitative PCR.

The present invention

1) separating the DNA from the honey sample and

2) provides a method for determining the authenticity of honey, comprising the step of detecting plant DNA and bee DNA from the separated DNA.

In the method of the present invention, 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). However, the method of separating DNA is not limited thereto.

In the method of the present invention, the process of detecting plant DNA and bee DNA in step 2) may be performed in any order or simultaneously.

In one embodiment of the present invention, the detection of step 2) may be performed using quantitative PCR. At this time, 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. However, 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.

If both the plant and bee DNA contents of honey are low, there are four possibilities. I. Possibility of impurity added honey (rice syrup or corn syrup is also derived from plants but contains little plant DNA through processing) ii. The possibility of microfiltering in the manufacturing process, and iii. The possibility of honey being treated at high temperatures above 90 ° C. during the manufacturing process, and iv. Pure honey is diluted with some or more impurities. Considering the fact that microfiltering is technically complicated and expensive and very unlikely to be used for the production of commercial honey products, it is unlikely that ii. Therefore, if the honey content of both the plant DNA and the bee DNA is low, it can be determined that the honey is impurity-added honey or honey treated at a high temperature of 90 ° C or higher.

If honey has a low plant DNA content but a high bee DNA content, the honey may be determined to be honeycomb honey. Sugar contains trace amounts of plant DNA, and sugar-supplied honeybees visit flowers very low.

If the honey content of the plant DNA is high but the bee DNA is 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.

If the honey content of the plant DNA and the bee DNA is low at the same time, 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.

In one embodiment of the method of the present invention, the honey sample in step 1) is 1 to 3 ml, the separated DNA in step 2) is 5 to 10 μl 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.

As an embodiment of the present invention, when the Ct value for the plant DNA is detected to be 27 or more under the quantitative PCR conditions, 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.

As another embodiment of the present invention, when the Ct value for honeybee DNA is detected at 25 or more under the quantitative PCR conditions, 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.

In the method of the present invention, when both the plant DNA and the bee DNA are not detected, 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.

In another embodiment of the present invention, when the Ct value for plant DNA is detected to be 27 or more under the quantitative PCR condition, but the Ct value for bee DNA is detected to be less than 25, a method for discriminating the honey sample as the specification honey do.

As another embodiment of the present invention, when the Ct value for plant DNA is detected to be 27 or more and the Ct value for bee DNA to be 25 or more in the quantitative PCR condition, the honey sample is determined as diluted honey. To provide.

In the method of the present invention, 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.

In the method of the present invention, 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.

As described above, 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.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

The reagents and solvents mentioned below were purchased from Sigma Aldrich ^® unless otherwise noted.

Example 1 Authenticity of Honey Using Detection of Plant and Bee DNA

1-1. Preparation of Honey Samples

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.

In the same area as above, the 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.

In the same area as above, 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.

1-2. DNA extraction

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. After the supernatant was completely removed, the remaining solids were vortexed for about 1 minute, and then 0.6 ml of the solution of Geno-Honey DNA kit and 40 μl of magnetic beads were added and vortexed for 10 seconds. After 2 minutes the microtubes were transferred to a magnetic stand (Jinfinity Bioscience, Augusta, USA). The supernatant was discarded and 1 ml of 70% ethanol was added and inverted and mixed 5-6 times on a magnetic stand, after which the supernatant was discarded again. It was then centrifuged at 14,000 rpm for 10 seconds and all liquid was removed by pipetting. Then, 50 μl of distilled water was added and pulse vortexed to extract DNA bound to the magnetic beads.

1-3. Detection of Plant and Bee DNA

Considering that honey contains various bee DNA and plant DNA, a universal primer for each was used as follows. The sequence of the primer is shown in Table 1 below.

TABLE 1

5 μl of the DNA extraction solution prepared in 1-2, 12.5 ul of 2 X SYBR premix Ex Taq (Takara, Japan) and 0.2 pM of each primer were used for quantitative PCR using a total of 25 ul. For quantitative PCR, Smartcyler (Cepheid, US) was used to perform quantitative pcr. Bee DNA measurements were performed for a total of 35 cycles so that annealing and extension occurred at 57 ° C. for 20 seconds after denaturation at 95 ° C. for 5 seconds after treatment at 95 ° C. for 30 seconds. Plant DNA measurements were performed for a total of 35 cycles so that annealing and extension occurred at 55 ° C. for 20 seconds after denaturation at 95 ° C. for 5 seconds after treatment at 95 ° C. for 30 seconds.

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.

TABLE 2

As shown in Table 2, in the case of specification honey, the Ct value of plant DNA was higher than that of acacia honey or miscellaneous honey, while the Ct value of bee DNA was similar, indicating that the plant DNA content was low and the bee DNA content was different from that of pure honey. I could see that there is no. Neither plant DNA nor bee DNA was detected in rice syrup and corn syrup.

Example 2. Confirmation of change of DNA content by heat treatment

1.5 ml of three-fold diluted acacia honey samples were transferred to microtubes and heat treated by placing them in a heat block set at 90 ° C., 80 ° C. and 55 ° C., respectively. Thereafter, the pellets are collected by centrifugation of the microtubes, and the pellets collected from two identical samples are combined, and then the same procedure as in Examples 1-2 and 1-3 is performed to obtain honeybee DNA and plant DNA from the heat-treated sample. The change in Ct value for was confirmed.

The results are shown in FIG. In the diagram at the top of FIG. 1, it can be seen that both honey bee DNA and plant DNA in honey treated at 90 ° C. were significantly reduced within 1 hour of heat treatment. In the diagram of FIG. 1, it can be seen that in the honey treated at 80 ° C., the plant DNA was maintained at about 80% after 2 hours of heat treatment, but the bee DNA decreased to less than 5% of the original DNA amount within 2 hours of heat treatment. . This seems to be because plant DNA protected by cell walls is more heat resistant than honey bee DNA. In the diagram at the bottom of Figure 1 it can be seen that in the honey treated at 55 ℃ both bee DNA and plant DNA almost no change.

From the above results, if neither bee DNA nor plant DNA is detected from the honey sample or only a small amount is detected, it may be heat treated at a temperature of 90 ° C. or higher during the manufacturing process. It can be judged that there is a possibility that the heat treatment.

Example 3. Identification of DNA After Long-Term Storage

To find out how bee 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. After extracting DNA according to the method described in Example 1, honeybee DNA and plant DNA were detected using quantitative PCR.

The results are shown in FIG. In Figure 2, both the K12 sample (top diagram) and the K17 sample (bottom diagram) were confirmed that the bee DNA and plant DNA contents were maintained constant over time.

From the above results, it was confirmed that 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.

Example 4 Identification of Commercial Honey

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.

TABLE 3

As shown in Table 3, only six samples (K4, K10, K12, K16, K17, and K22) of the 23 domestic honey product samples contained high levels of plant DNA. Two samples (K11 and K19) did not contain any plant DNA. As such, samples containing little or no plant DNA may be considered honey or impurity added honey or honey processed at a high temperature of 90 ° C. or higher during the manufacturing process. In case of K6, 7, 8, 13, 14, 20, each DNA is contained but the value is high, so pure honey can be judged as honey diluted with impurities. In some cases (K2, 3, 5, 18) it is considered to be honey bee due to plant DNA deficiency. In addition, the four samples (K9, 11, 19, and 21) do not contain any pure DNA or contain trace amounts of the two DNAs, which do not contain pure honey (i.e., are made only of impurities) or It can be considered processed honey.

Some products listed the 13C / 12C isotope ratio on the label according to the honey autolabel. K1, 2, 6, 13, and 14 samples contained very small amounts of plant DNA, although the 13C / 12C isotope ratio was described as 23.5. This suggests that the products may have errors in the 13C / 12C isotope ratio on the label, or added C4 plants such as beet.

In addition, 14 kinds of imported honey products and 2 kinds of honey products of unknown country were purchased from a distributor of Paengoroa.New Zealand or a department store in Seoul, and quantitative PCR was used as described in Example 1 above. Bee DNA and plant DNA included were measured. Imported honey products do not have a 13C / 12C isotope ratio on the label, so there is no data on this. Specific data are shown in Table 4 below. The Ct value is the average value obtained after two independent analyzes.

TABLE 4

As shown in Table 4, six samples (I2, 10, 11, 12, 13) are judged to be good quality products containing a large amount of two DNA. Samples of (I1, 3, 6, 7, 8, and 9) are judged to be honey because of their significantly lower plant DNA content. The I4 sample was found to be free of pure honey because of its lack of two DNAs. Two samples of unknown origin (U1, U2) also contain very small amounts of plant DNA and bee DNA, which is believed to be diluted honey.

Claims (10)

1. A method for determining the authenticity of honey samples by detecting plant DNA and bee DNA in honey samples.
2. The method of claim 1, wherein the detection of plant DNA and bee DNA is performed using quantitative PCR.
3. 1) separating the DNA from the honey sample; and

   2) detecting the authenticity of the honey sample, comprising the step of detecting plant DNA and bee DNA from the separated DNA.
4. The method of claim 3, wherein step 2) is performed using a plant common primer pair of SEQ ID NO: 1 and SEQ ID NO: 2 and a bee common primer pair of SEQ ID NO: 3 and 4.
5. The method of claim 3, wherein the honey sample in step 1) is 1 to 3 ml, and in step 2), the separated DNA is 5-10 μl of the DNA solution isolated from the honey sample and the detection is SEQ ID NO: 1 and the sequence. A method performed by quantitative PCR using a plant consensus primer pair of No. 2 and a bee consensus primer pair of SEQ ID NOs: 3 and 4.
6. The method according to claim 5, wherein when the Ct value of the plant DNA is detected to be 27 or more, the honey sample is determined as a sample containing no honey, specification honey, diluted honey, or pure honey, which is heat-treated at 90 ° C or higher. Way.
7. The method according to claim 5, wherein when the Ct value of the honeybee DNA is detected to be 25 or more, the honey sample is determined as a sample containing no honey, specification honey, diluted honey, or pure honey at least 80 ° C. Way.
8. 4. The method according to claim 3, wherein when both plant DNA and bee DNA are not detected, the honey sample is determined as a sample containing no honey or pure honey heat-treated at 90 ° C or higher.
9. The method according to claim 5, wherein when the Ct value for the plant DNA is detected to be 27 or more but the Ct value for the bee DNA is to be detected to be less than 25, the honey sample is determined as the specification honey.
10. The method of claim 5, wherein when the Ct value for plant DNA is detected to be 27 or more and the Ct value for bee DNA is detected to be 25 or more, the honey sample is determined as diluted honey.

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