KR20160137203A - Method for Adulteration of Pure Honey with Rice Syrup - Google Patents
Method for Adulteration of Pure Honey with Rice Syrup Download PDFInfo
- Publication number
- KR20160137203A KR20160137203A KR1020150071995A KR20150071995A KR20160137203A KR 20160137203 A KR20160137203 A KR 20160137203A KR 1020150071995 A KR1020150071995 A KR 1020150071995A KR 20150071995 A KR20150071995 A KR 20150071995A KR 20160137203 A KR20160137203 A KR 20160137203A
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- South Korea
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- honey
- additive
- natural honey
- primer set
- detecting
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
Abstract
The present invention relates to a primer set for detection of an additive in natural honey having a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, and a method for detecting an additive mixed with natural honey by performing an enzyme polymerization reaction (Polyemrase Chain Reaction And a kit for detecting an additive in natural honey comprising the primer set.
The primer set and detection method for detecting additives in natural honey according to the present invention can be easily and quickly carried out by mixing rice syrup in natural honey without expensive equipments required for conventional carbon isotope analysis method used for honey quality inspection .
Description
This technique relates to a method for distinguishing fake honey mixed with additives such as rice syrup (phytate).
Honey is a honey bee collecting honey from honey and storing it in a honeycomb. It means not adding any additives such as firearm, royal jelly, saccharides, sweeteners and so on after soaking (FOOD STANDARDS, KOREA FOOD INDUSTRY ASSOCIATION). Honey contains not only ideal nutritional components such as pollen, vitamins, proteins, minerals, and amino acids but also sugar components such as glucose and fructose, preventing and treating fatigue, anemia, diabetes and liver disease, eliminating hangovers, And promotion of pediatric development. Fructose, which is a main component of honey, has a higher absorption rate than sucrose and has a high sweetening effect, and thus is consumed in a large amount in the food industry as a sweetener.
Honey is an unusual color and aroma depending on the kind of plant. It is generally grown in plums such as plums, peaches, cherries, cherries, chestnuts, clover, citrus, sari, apple, acacia, chestnut, persimmon, Name it after the name of honey.
In addition, honey can be classified into Extracted honey and Comb honey depending on the production method. Honey separated by centrifugal force or compression is called separation wheat, and liquid, crystal, cream It can be divided and stored for consumption. Honey is produced in a certain size as it is produced without separating the honey that has been sealed. Honey has been widely used as a health food such as medicines and nutrients in addition to being widely used as a sweetener and the like.
Honey has been a problem for a long time because there is an example of mixing honey with sugar or syrup, and therefore various studies have been conducted for authenticity or quality evaluation. As part of this problem, as of August 1, 2009, the Korea Food and Drug Administration (KFDA) has designated honey as a specification honey to distinguish honey from pure honey. , And started to run an 'autonomous marking system' that displays the result of carbon isotope analysis together with the mixing ratio of pure honey and specimen honey.
Conventionally, various chemical analysis methods have been mainly used for quality evaluation and authenticity discrimination of honey. In consideration of diversity of honey, taxonomic classification of bees, seasonal factors and regional difference, the quality of honey is evaluated by one chemical analysis method And accuracy of authenticity discrimination. The carbon isotope analysis method, which is a typical analysis method, compares the carbon isotope ratios of C3 plants and C4 plants in food, and it is the origin of its origin. The C3 plant group is the most temperate plant group, the C4 plant group is corn, These include plants that grow in hot and dry areas such as sorghum. Plant groups belonging to these two groups accumulate in the plant body at different ratios of 12C and 13C, so the ratio can be checked to determine the origin.
However, such an analysis method requires many expensive equipments, complexity of analytical methods, continuous management of analytical instruments, high cost and long analysis time, as well as various disadvantages such as involving structural destruction at the level of molecular structure It implies.
Therefore, the present inventors have made intensive efforts to develop a simple and sensitive method for judging whether rice syrup is mixed in honey. As a result, it has been found that by using a primer set capable of detecting rice-derived DNA in honey, chain reaction, PCR). As a result, it was confirmed that the mixture of rice syrup in honey could be easily distinguished and the present invention was completed.
It is an object of the present invention to provide a primer set for detecting an additive in natural honey having a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, a method for detecting an additive in natural honey using the same and a kit thereof.
In order to achieve the above object, the present invention provides a primer set for detecting an additive in natural honey having a nucleic acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
The present invention also provides a method for detecting an additive mixed with natural honey by performing an enzyme polymerization reaction (Polymerase Chain Reaction (PCR)) using a primer set having a nucleic acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:
The present invention also provides a kit for detecting an additive in natural honey comprising a primer set having a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.
The primer set and detection method for detecting additives in natural honey according to the present invention can be easily and quickly carried out by mixing rice syrup in natural honey without expensive equipments required for conventional carbon isotope analysis method used for honey quality inspection .
1 is a PCR result of confirming the detection of a cyc of honey including rice syrup (M: Marker; 1: natural honey; 2: 5% rice syrup in honey; 3: 10% rice syrup in honey: 4: rice syrup) .
Hereinafter, the present invention will be described in detail.
Some of the honey that is imported or distributed in Korea is a problem because it is a fake honey which is costly and less expensive than the natural honey and the 'carbon isotope analysis method', which is generally used as a honey quality evaluation method, Is required.
The present inventors have made intensive efforts to develop a technique for quickly distinguishing honey added with rice syrup (phytate). As a result, they have found that when DNA extracted from honey is used as a template and a specific nucleic acid sequence is used as a template, And the present invention has been completed.
In one aspect, the present invention relates to a primer set for detection of an additive in natural honey having a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.
The primer set of the present invention is hybridized to the cyc region of the cytochrome C gene of rice and has the ability to detect the region.
Each primer of the present invention may be labeled radioactive or non-radioactive and the non-radioactive label may be labeled with conventional biotin, Dig (digoxigenin), FRET (fluorescence resonance energy transfer) Cy5, Cy3, etc.), but is not limited thereto. In addition, the primer can be used as a probe.
In the present invention, the natural honey is characterized in that the natural honey is originated from a mulberry plant selected from the group consisting of plum, peach, cherry, cherry, walnut, clover, citrus, sari, apple, acacia, chestnut, persimmon, buckwheat and rapeseed , But is not limited thereto.
In the present invention, the additive is characterized by being rice syrup or whey protein.
It is a natural sweetener made by cutting the grain into maltose and cutting it like honey, and it is called as 'artificial honey' because it is called 'the honey of natural life' because it is artificial honey. Jochong is made by fermentation of malt or enzyme with starch such as rice, sorghum, millet, and corn as a raw material. It is usually produced through an immersion step, a fermentation step, a saccharification step, a filtration and purification step, and a concentration step.
The additives in the present invention are preferably characterized by being rice cakes made of rice as a raw material.
In another aspect, the present invention relates to a method for detecting an additive mixed with natural honey by performing an enzyme polymerization reaction (Polymerase Chain Reaction (PCR)) using a primer set having a nucleic acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 .
In another aspect, the present invention relates to a kit for detecting an additive in natural honey comprising a primer set having a nucleic acid sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2.
More particularly, the present invention relates to a method for detecting DNA of a honey mixed with an additive, and a primer set capable of amplifying the cyc region of the cytochrome C gene of rice with the separated DNA (PCR), and analyzing the amplified reaction product by electrophoresis. The method of analyzing the reaction product may be performed by electrophoresis But is not limited thereto.
The polymerase chain reaction (PCR) of the present invention can be carried out by conventional PCR, but also by hot-start PCR, nested PCR, multiplex PCR, reverse-transcriptase PCR, degenerate oligonucleotide primer (DOP) Modified PCR reactions such as PCR, In-Situ PCR, Micro PCR, or Lab-on-a-chip PCR reactions may be used.
In the present invention, the natural honey is characterized in that the natural honey is originated from a mulberry plant selected from the group consisting of plum, peach, cherry, cherry, walnut, clover, citrus, sari, apple, acacia, chestnut, persimmon, buckwheat and rapeseed , But is not limited thereto.
In the present invention, the additive is characterized by being rice syrup or whey protein.
It is a natural sweetener made by cutting the grain into maltose and cutting it like honey, and it is called as 'artificial honey' because it is called 'the honey of natural life' because it is artificial honey. Jochong is made by fermentation of malt or enzyme with starch such as rice, sorghum, millet, and corn as a raw material. It is usually produced through an immersion step, a fermentation step, a saccharification step, a filtration and purification step, and a concentration step.
The additives in the present invention are preferably characterized by being rice cakes made of rice as a raw material.
The enzyme polymerization reaction (PCR) used in the detection method of the present invention is not particularly limited, and may be one of various known improvement methods. For example, reagents such as Tris-HCl, KCl, MgCl 2 , dNTP, and Taq DNA polymerase in addition to a primer set and a DNA template are mixed to prepare a PCR reaction solution. One cycle of PCR consists of three steps of heat denaturation, annealing of primer, and DNA synthesis reaction by DNA polymerase. Since each step requires different reaction temperature and reaction time in different cases , The nucleotide sequence of the DNA region to be amplified and the length thereof, can be easily selected by those skilled in the art.
PCR products can be identified using an immunological method, but the test substance can be identified using a positive or negative control by electrophoresis.
In addition, the kit for detecting an additive in natural honey containing the primer set of the present invention can be used in a detection method by PCR. Specifically, the reagents included in the kit include dNTP, MgCl 2 , Taq polymerase, Tris-HCl, glycerol, DMSO, DNA for positive control, DNA for negative control, and distilled water. The above reagents may be packaged independently in a kit, or may be provided in the form of a mixture of two or more kinds of reagents. There is no particular limitation on the concentration of each reagent contained in the kit, but preferably the PCR of the present invention is carried out For example. In the kit of the present invention, only the information on the preferable PCR condition may be attached or may include only the primer set.
The present invention will be described in more detail with reference to the following examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited by these examples in any sense.
In this case, unless otherwise defined, technical terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the following description and the accompanying drawings, A description of known functions and configurations that may unnecessarily obscure the description of the present invention will be omitted.
Example 1: Extraction of DNA from honey
To 10 g of each of a honey sample or a rice syrup 100% sample mixed with 5% or 10% of rice syrup to natural or natural honey, 20 ml of sterilized water was mixed and diluted, and the mixture was centrifuged at 4000 rpm for 10 minutes ), From which the supernatant was removed and the resulting precipitate was obtained. The precipitate was extracted with a genomic DNA extraction kit (Bioneer, Korea) according to the manufacturer's recommended method and used as a template for PCR.
Example 2: Confirmation of Rice Syrup Mixing by PCR
To 10 ng of DNA extracted from honey, 1 占 퐇 of 10 mM dNTP; 2X of 10X PCR buffer and 0.5 Unit Taq DNA polymerase (Bioneer, Korea) were mixed, and the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 were mixed at a concentration of 5 μM to a final volume of 20 μL .
PCR reaction conditions were 95 C for 10 minutes, followed by amplification of 40 cycles of PCR (Takara, Japan) using 95 cycles of 30 seconds, 60 seconds and 30 seconds, and 72 seconds and 30 seconds as one cycle. Finally, the reaction mixture was maintained at 72 ° C for 7 minutes and then at 4 ° C. The PCR reaction product obtained by the above reaction was subjected to electrophoresis using 1.2% agarose gel (2% E-Ge1, Invitrogen) containing ETBR (ethidium bromide) to determine the presence or absence of the rice gene in the sample Respectively.
The primers used in the PCR were selected from the DAN sequences of the rice genome used to distinguish genetically modified (GMO) rice.
SEQ ID NO: 1: 5'-GCACTGGGCGCACTTGGTCT-3 '
SEQ ID NO: 2: 5'-TTCGCCTCTTCTTCCTCCTCTCG-3 '
As a result, no band was observed in the sample derived from the natural honey, but a sample containing rice syrup was confirmed to have a band of 121 bp, and in particular, it was detected that the concentration of rice syrup was 5% (by weight) )
<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Method for Adulteration of Pure Honey with Rice Syrup <130> P2015-0050 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 1 gcactgggcg cacttggtct 20 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 2 ttcgcctctt cttcctcctc tcg 23
Claims (10)
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| KR1020150071995A KR101747243B1 (en) | 2015-05-22 | 2015-05-22 | Method for Adulteration of Pure Honey with Rice Syrup |
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| KR1020150071995A KR101747243B1 (en) | 2015-05-22 | 2015-05-22 | Method for Adulteration of Pure Honey with Rice Syrup |
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| KR20160137203A true KR20160137203A (en) | 2016-11-30 |
| KR101747243B1 KR101747243B1 (en) | 2017-06-14 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107449837A (en) * | 2017-01-09 | 2017-12-08 | 北京工商大学 | Differentiate honey and syrup using non-saccharide property material |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014171767A1 (en) * | 2013-04-17 | 2014-10-23 | 주식회사 제놀루션 | Method for determining authenticity of honey using dna detection |
| KR101444155B1 (en) | 2014-05-16 | 2014-09-26 | 대한민국 | Primer set for determining incorporation of wheat in rice-processing foods, method of determining incorporation of wheat in rice-processing foods using the same, and kit comprising the same |
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- 2015-05-22 KR KR1020150071995A patent/KR101747243B1/en active IP Right Grant
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107449837A (en) * | 2017-01-09 | 2017-12-08 | 北京工商大学 | Differentiate honey and syrup using non-saccharide property material |
| CN107449837B (en) * | 2017-01-09 | 2020-10-30 | 北京工商大学 | Method for identifying honey and syrup by using non-sugar characteristic substances |
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| KR101747243B1 (en) | 2017-06-14 |
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