KR20130014713A - A method for rapid detecting mycotoxins in fermented tea - Google Patents

Abstract

PURPOSE: A method for quickly and simply analyzing mycotoxin in a tea is provided to conduct usual tests without expensive equipment and to improve the reliability of enzyme immunoassay using an ELISA kit. CONSTITUTION: A method for analyzing mycotoxin in a tea comprises: a step of preparing a solvent extract from tea leaves using one or more kinds of alcohols selected from C1-C5 alcohols; a step of eluting the solvent extract using 70% methanol solution and purifying the extract; and a step of measuring mycotoxin by enzyme immunoassay. The mycotoxin is aflatoxin or ochratoxin A. The enzyme immunoassay comprises a step of performing ELISA(Enzyme-Linked Immunosorbent Assay) using each antibody for mycotoxin. [Reference numerals] (AA) Extract of 70% microorganism fermented tea C18

Description

Rapid Analysis of Fungal Toxins in Microbial Fermented Teas {A METHOD FOR RAPID DETECTING MYCOTOXINS IN FERMENTED TEA}

The present invention relates to a method for quickly and simply analyzing fungal toxins that may remain in a microbial fermentation tea.

Tea is one of the world's three favorite beverages, and humans have been drinking it since ancient times. Tea in the broadest sense means that the young shoots, leaves, flowers, stems, roots, fruits, etc. of plants are processed and manufactured as raw materials, and mainly refers to the favorite foods which leach the raw materials into water and drink the filtrate. Narrow tea is made by processing the leaves of Camellia senensis , which belongs to Camellia.

In Korea, the wider meaning of tea in food industry means leach teas among teas, and narrow teas are defined as single leach teas. According to the food industry, the single leach tea is divided into green tea, oolong tea, black tea, processed grain tea and other single leach tea.

In Korea, with respect to tea, mainly green tea, oolong tea and black tea, the classification of the green tea, oolong tea and black tea is mainly divided according to the degree of fermentation, the green tea means tea that does not ferment, the oolong tea It means semi-fermented, the black tea means fully fermented. In China, tea is classified according to the degree of oxidation of polyphenols contained in tea leaves, and is classified into six types such as green tea, yellow tea, black tea, blue tea, white tea and black tea. In Japan, tea leaves are classified into non-fermented tea, weakly fermented tea, semi-fermented tea, fully fermented tea, post-fermented tea (microbial fermented tea), and reprocessed tea.

Oxidation of polyphenols of unfermented tea, semi-fermented tea, and pre-fermented tea such as green tea, oolong tea, and black tea, which are mainly consumed among the above-mentioned teas, is due to the action of various enzymes originally present on tea leaves, not by fermentation by microorganisms. This is not a fermentation in the exact sense. In this respect, only the fermented tea such as black tea can be said to be a fermented tea that has undergone the fermentation process by microorganisms such as mold in a strict sense.

Recently, with increasing interest in health, consumption of various teas has been increasing rapidly. Among the teas, the consumption of which is rapidly increasing is well known as Pu-erh tea, and the above-mentioned microbial fermented tea is also called black tea or post-fermented tea.

The tea is included in the fermented tea or microbial fermented tea fermented by the microorganisms, not the enzyme of tea leaves in the classification of tea, refers to a fermented tea of relatively low fermentation degree manufactured in Yunnan province of China, but currently in Korea Consumers call all microbial fermented teas a boy tea.

The microbial fermented tea has a history of about 3,000 years, and was mainly produced in the south of China, Sichuan, Yunnan, Hunan, Hubei or Guizhou, and was also consumed in areas such as Tibet or Mongolia, which is north of China.

The microbial fermented tea was mainly manufactured by inactivating enzymes immediately after harvesting the mesenchymal leaves, followed by sterilization, molding, and drying after fermentation using microbial spores after the process of keeping in mind and drying. The microorganisms involved in fermentation of the microbial fermented tea are mainly known as fungi such as Aspergillus sp., Rhizopus sp. Or Penicillium sp. It is known that the position of the dominant species varies depending on the processing process and environmental conditions.

Since the microorganism fermented tea is a long time from several microorganisms grow during the fermentation process and the fermentation period is short, about 2 years to several decades, the microbial fermented tea is a fungal toxin (Mycotoxin) which is a harmful component that can be produced by these microorganisms. Is always exposed to the possibility of In particular, Aflatoxin and Ochratoxin A, which are produced as a secondary metabolite by fungi such as Aspergillus sp. Or Penicillium sp. have. In recent years, in the tea industry, not only green tea from primary processing but also black tea, oolong tea, blue tea and the above mentioned process for preparing Hazard Analysis Critical Control Point (HACCP) for the entire process of manufacturing secondary processed foods using these teas. As demands increase, it is time to monitor hazards about the possibility of mold toxin contamination in the storage and distribution of tea raw materials.

The fungal toxin is regulated by setting standards in each country for inclusion in food. The aflatoxin is regulated by setting a limit of 0 to 50 ppb in a total amount (B1 + B2 + G1 + G2) in about 50 countries around the world. For example, the US regulates all foods in the range of 20 ppb, Australia and Canada regulate the range of 15 ppb, especially nuts, and the EU regulates 4 ppb to 15 for grains and nuts. Regulated in the ppb range. In Korea, grains, soybeans, peanuts, nuts and simple processed products are regulated in the range of 15 ppb or less for total aflatoxin, 10 ppb or less for aflatoxin B1, and 5 ppb or less for olatoxin A. In the case of raw material feed is regulated in the range of 50 ppb or less, compounded feed in the range of 10 to 20 ppb or less.

The analytical method for the fungal toxin has been steadily developed since the association between the chemicals produced in the fungus and the diseases caused in the animals has been steadily developed.

The widely used methods for detecting the fungal toxin include a detection method using a chromatographic method such as thin layer chromatography (TLC) and a detection method using an enzyme-linked immunosorbent assay (ELISA).

The detection method using the thin layer chromatography method was a method used to define a fungal toxin as a chemical for the first time, but was mainly used in the past, but recently, high performance liquid chromatography (HPLC) has been developed to develop a high performance liquid chromatography. It is being replaced by the detection method by chromatography. The analysis method using the high performance liquid chromatography takes a long time in the extraction and purification process of the sample, there is a problem that expensive equipment and skilled technicians are required.

In addition, the enzyme immunoassay method has been applied to the detection method of the fungal toxin as the antibody production against the fungal toxin was possible in the 1970s and 1980s, and it is relatively inexpensive, and recently, the verification of the American Association of Analytical Chemists (AOAC) ELISA kits for fungal toxin analysis received are commercially available. However, since most of the fungal toxin is a low molecular weight substance having an average molecular weight of 1,000 Daltons or less, a problem has been pointed out that detection is limited for various fungal toxins.

The immunoassay used to detect the fungal toxin is mostly a competitive assay, in which a direct assay is performed by attaching an antibody specific to the fungal toxin and a fungal toxin or a mycotoxin-protein conjugate instead of the antibody to the surface. There is an indirect analysis method that is attached and analyzed. The immunoassay is mainly an enzyme immunoassay, and in the case of the enzyme immunoassay, it can be always analyzed in a laboratory, and the labor and cost can be reduced, thereby making it a simpler and more efficient detection technology.

Due to these advantages, to date, in relation to the analysis of fungal toxins, assays using immunoassays for total aflatoxin and okratoxin A, specifically, assays using an ELISA kit (Enzyme-linked immunosorbent assay kit), are used as food, specifically grains. , Nuts or milk or dairy products. However, there have been no reports on the analysis of fungal toxins of microbial fermented teas using immunoassay or enzyme immunoassay, such as ELISA kit. Due to the characteristics of tea samples rich in various polyphenols and low-molecular substances, there is a large amount of factors that interfere with the enzyme immune reaction between the antibody and fungal toxins against fungal toxins. This is because there is a difficulty in analyzing by using a device capable of doing so.

In the past, immunoassay methods were mainly used in fields related to disease diagnosis. For example, in Korean Patent Application Publication No. 10-2011-0077635 (published on July 7, 2011), a multi-marker composed of an autoantibody marker for diagnosing breast cancer and a combination thereof is used. A diagnostic kit is described, and Republic of Korea Patent Publication No. 10-2011-0077636 (published Jul. 7, 2011) discloses a large intestine comprising a combination of protein marker defensin 5 and ROD 1 protein for colon cancer prognosis and an antibody against each of them. The cancer prognosis diagnosis kit is described, and Korean Patent Publication No. 10-2002-0032752 (published on May 4, 2002) discloses a method for simultaneously detecting various types of target substances through multiple immune responses, and a kit for detecting the same. It is. However, due to the difficulty of the above analysis, a method for confirming microbial toxins through immunoassay for the management of a variety of microorganisms including fermented tea has not been reported yet, the prior literature has not been searched.

In order to solve the problems of the prior art, it is an object of the present invention to provide an analysis method capable of quickly and simply analyzing microbial toxins with a simple and high level of accuracy for risk management of microbial fermented tea.

In order to achieve the above object, the present invention provides an analytical method capable of analyzing microbial toxins from the car with a simple and high level of accuracy.

Specifically, expensive equipment is not required in the past, and a simple kit can always be analyzed in the laboratory, minimizes labor and cost, and uses a simple ELISA kit (Enzyme-linked immunosorbent assay kit), The present invention provides a method for the analysis of fungal toxins from tea that can maintain high levels of accuracy by effectively removing the factors that interfere with the enzyme immune reaction between the antibodies and the fungal toxins.

The present invention comprises the steps of preparing a solvent extract using the tea leaves selected from the group consisting of water and alcohols having 1 to 5 carbon atoms; Preparing a purified product by eluting a 70% methanol aqueous solution with an elution solvent using a Sep-Pak C18 cartridge; And it relates to a method for analyzing the fungal toxin from the tea comprising the step of measuring the fungal toxin (Mycotoxin) from the purified by enzyme immunoassay.

The tea may be any one selected from the group consisting of microbial fermented tea, green tea, blue tea, black tea, oolong tea, and mixtures thereof, and may be a microbial fermented tea, which is preferably produced through a fermentation process by microorganisms.

In this aspect, the present invention comprises the steps of preparing a solvent extract using the tea leaves of the microbial fermented tea selected from the group consisting of water and alcohols having 1 to 5 carbon atoms; Preparing a purified product by eluting a 70% methanol aqueous solution with an elution solvent using a Sep-Pak C18 cartridge; And it relates to a method for analyzing the fungal toxin from the microbial fermentation tea comprising measuring the fungal toxin (Mycotoxin) from the purified by enzyme immunoassay.

In addition, the fungal toxin may be any one selected from the group consisting of Aflatoxin and Ochratoxin A.

In addition, the solvent of the step of preparing the solvent extract may be any one selected from the group consisting of water, methanol and mixtures thereof.

In addition, the enzyme immunoassay may be performed using an antibody against any one fungal toxin selected from the group consisting of Aflatoxin and Ochratoxin A. The enzyme immunoassay may be a method of performing an ELISA (Enzyme-linked immunosorbent assay) using an antibody against any one fungal toxin selected from the group consisting of Aflatoxin and Ochratoxin A.

Unlike other foods that previously used enzyme-based immunoassay such as ELISA to analyze fungal toxins, polysaccharides such as green tea and microbial fermented tea, that is, teas, are used as ELISA measurement method due to the interference effect of ingredients contained in tea leaves. It was impossible to detect fungal toxins effectively.

The inventors of the present invention endeavored to solve such a problem, and as a result, the interference effect when performing ELISA using an antibody against a fungal toxin such as aflatoxin or okratoxin A is various polyphenols and low molecular weight substances contained in tea leaves. Increased error by inhibiting the binding between an antibody to a fungal toxin, such as aflatoxin or okratoxin A, or by inaccurately binding a low-molecular weight non-toxic substance such as aflatoxin or okratoxin A to the antibody. It was confirmed that. Based on these research results, while solving the same problems of the existing method using the ELISA measuring device, while studying the analysis method that can replace the current high performance liquid chromatography (HPLC) method, After extracting the microbial fermentation tea using a 70% methanol aqueous solution solvent to prepare a solvent extract, when the solvent extract is eluted in a 50% methanol aqueous solution using a Sep-Pak C18 cartridge, enzyme immunoassay It was confirmed that most of the interference factor was removed, most of the fungal toxin was eluted in 70% aqueous methanol solution to complete the present invention.

Hereinafter, the present invention will be described in more detail.

In the present invention, the tea leaves are separated from the filtrate prepared by leaching tea into water for drinking, and the leaves of the tea tree, ie, leaves collected from the tea tree before leaching into water, such as apricot, mind or dry The processed or leaves collected from the tea tree or processed leaves obtained by the tea tree means processed through a fermentation process such as enzyme fermentation or microbial fermentation. Examples of the tea include green tea, green tea, black tea, oolong tea, and microbial fermented tea.

In the present invention, microbial fermented tea means a fermented tea fermented by a microorganism in comparison with an enzyme fermented tea such as black tea fermented by enzymes of tea leaves among fermented teas, and is also called a post-fermented tea or black tea. An example of the microbial fermentation tea is Pu-erh tea. The microbial fermented tea is a fermentation ripening step of fermenting tea leaves, for example, a teal step to harvest and then inactivate the enzyme by heating and then inactivating enzymes, a step of rubbing the fresh tea leaves, drying the dried tea leaves and fermenting the dried tea leaves using microorganisms. It may be prepared by a method comprising the step.

In the present invention, immunoassay (immunoassay) means an assay that can test the target material using the specific binding ability of the antibody to the target material to be measured with the enzyme or isotope labeled antigen. The immunoassay includes enzyme immunoassay and fluorescence immunoassay.

The enzyme immunoassay (EIA) is a method of labeling an antibody with an enzyme and detecting a substance to which the antibody binds, also called an enzyme immunoassay. It is distinguished from radioimmunoassay using antibodies labeled with radioisotopes or fluorescence immunoassay using antibodies labeled with fluorescent substances. The principle of the enzyme immunoassay is to detect an antibody (specific antibody) that has reacted with the antigen to be measured as an antibody (labeled antibody) by chemically binding an enzyme such as peroxidase or galactosidase to the antibody. In this way, not only qualitative but also quantitative analysis is possible. The enzyme immunoassay method has a direct method and an indirect method. In the case of the direct method, the enzyme-labeled antibody and the antigen are separated from each other, and then, when the enzyme is activated, a substrate that develops color is added to optically measure the amount of degradation thereof. The amount of bound antibody is calculated by calculating the amount of bound antibody, and the indirect method is a method of immobilizing the antigen to be detected on a plate or the like, adding a specific antibody and washing, adding a labeled antigen, and reacting the enzyme of the bound label antigen with a colorant to quantify it. Anti-antibody (secondary antibody) is a method of determining the antigen or antibody amount in the antigen-antibody conjugate from the enzymatic activity of the secondary antibody.

Enzyme-linked immunosorbent assay (ELISA), which is typically used in the enzyme immunoassay method, is a method of measuring an antigen or an antibody by immobilizing it in a small groove of a glass bead or plastic plate and then contacting the sample.

The present invention comprises the steps of preparing a solvent extract using the tea leaves selected from the group consisting of water and alcohols having 1 to 5 carbon atoms; Purifying the solvent extract by SPE (Solid Phase Extraction) extraction to prepare a purified product; And it relates to a method for analyzing the fungal toxin from the tea comprising the step of measuring the fungal toxin (Mycotoxin) from the purified by enzyme immunoassay.

The tea may be any one selected from the group consisting of microbial fermented tea, green tea, green tea, black tea, oolong tea, and mixtures thereof, preferably microbial fermented tea or green tea, and more preferably microbial fermented tea. .

In this aspect, the present invention comprises the steps of preparing a solvent extract using the tea leaves of the microbial fermented tea selected from the group consisting of water and alcohols having 1 to 5 carbon atoms; Preparing a purified product by eluting a 70% methanol aqueous solution with an elution solvent using a Sep-Pak C18 cartridge; And it relates to a method for analyzing the fungal toxin from the microbial fermentation tea comprising the step of measuring the fungal toxin (Mycotoxin) from the purified by enzyme immunoassay.

The fungal toxin may be one or more selected from the group consisting of aflatoxin and okraatoxin A.

The preparing of the solvent extract may be performed by a method of preparing a conventional plant extract using a solvent for tea leaves, preferably microbial fermented tea or tea leaves of green tea, and more preferably tea leaves of microbial fermented tea. Can be. The solvent may be at least one selected from the group consisting of water and alcohols having 1 to 5 carbon atoms, preferably at least one selected from the group consisting of water, ethanol and methanol, more preferably 50% to 99 % Aqueous methanol solution. The preparing of the solvent extract may additionally perform a filtration process using filter paper after the extraction step.

The step of preparing the purified product was performed by solid phase extraction (SPE) extraction. The SPE extraction method refers to a method of purifying by extracting only a substance eluted to a specific solvent among substances adsorbed on the column using a Sep-Pak column. The column may preferably use a Sep-Pak cartridge filled with C18 filler. The C18 filler may be one containing a C ₁₈ H ₃₇ chain.

In this aspect, the SPE extraction method may be preferably performed by purifying the extract by eluting a 70% methanol aqueous solution with an elution solvent using a Sep-Pak C18 cartridge. .

More specifically, the step of preparing a purified product is the process of filling the Sep-Pak C18 cartridge (cartridge) the solvent extract; Injecting a 50% aqueous methanol solution into the Sep-Pak C18 cartridge (cartridge) filled with the solvent extract as an elution solvent to remove the eluate; And injecting 70% aqueous methanol solution into the elution solvent into a Sep-Pak C18 cartridge from which the 50% aqueous methanol solution eluate was removed.

More specifically, the step of preparing the purified product comprises the steps of filling the solvent extract in a Sep-Pak C18 cartridge (cartridge); Removing the eluate by injecting distilled water into the elution solvent in a Sep-Pak C18 cartridge filled with the solvent extract; Injecting a 20% aqueous methanol solution into the Sep-Pak C18 cartridge (cartridge) from which the distilled water eluate is removed as an elution solvent to remove the eluate; Injecting 50% aqueous methanol solution into the Sep-Pak C18 cartridge (cartridge) from which the 20% aqueous methanol solution eluate was removed as an elution solvent to remove the eluate; And injecting 70% aqueous methanol solution into the elution solvent into a Sep-Pak C18 cartridge from which the 50% aqueous methanol solution eluate was removed.

Filling the Sep-Pak C18 cartridge (cartridge) is a Sep-Pak C18 cartridge (Sep-Pak C18 cartridge activated by using one or more selected from the group consisting of distilled water, methanol and mixtures thereof It can be performed by adding a solvent extract to a cartridge) and concentrating with nitrogen.

The step of measuring the fungal toxin by enzyme immunoassay may be performed using an antibody specific for the fungal toxin. The fungal toxin may be at least one selected from the group consisting of Aflatoxin and Ochratoxin A. In this aspect, the antibody may be a specific antibody to aflatoxin or an antibody specific to okratoxin A.

The enzyme immunoassay method adds an antibody against any one fungal toxin selected from the group consisting of aflatoxin and olatoxin A to the purified product eluted with the aqueous 70% methanol solution, and induces an enzymatic reaction to the degree of enzymatic reaction. According to the method can be carried out by quantifying the content of the fungal toxin. For example, the purified product prepared by eluting with 70% aqueous methanol solution may be performed by ELISA (Enzyme-linked immunosorbent assay) using a specific antibody against aflatoxin or olatoxin A. The ELISA method can be performed using an ELISA kit (ELISA kit) for aflatoxin or okratoxin A previously disclosed.

The necessity of testing for the inclusion of microbial toxins in food is increasing the need for human health and well-being, the need to protect livestock from acute toxicity due to the mass production of livestock, and improving productivity through the defense of chronic exposure to fungal toxins. It can be sufficiently explained by the like.

In relation to such microbial toxins in foods, in particular, the present invention for the management of various harmful factors such as microorganism fermented tea and green tea, green tea, black tea, oolong tea, the demand of which is rapidly increasing due to the functional effect of the present invention, Microbial toxins can be remarkably improved by simple pretreatment using ELISA KIT. The economic effect is very great in that a method for qualitative and quantitative detection has been proposed.

Figure 1 is a graph showing the HPLC analysis results (chromatogram) for the total aflatoxin standard reagent, 70% methanol solution extract of green tea leaves and 70% methanol solution extract of microbial fermented tea according to an embodiment of the present invention, Figure 1a Results of aflatoxin standard reagent, Figure 1b is the result of 70% methanol green tea solution extract, Figure 1c is a result of 70% methanol extract of fermented tea microorganisms. In FIG. 1A, B1, B2, G1, and G2 mean aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2, respectively.
Figure 2 is a graph showing the HPLC analysis results (chromatogram) for the okratoxin A standard reagent, 70% methanol solution extract of green tea leaves and 70% methanol solution extract of microbial fermented tea according to an embodiment of the present invention, Figure 2a Figure 2b is the result of the okratoxin A standard reagent, Figure 2b is the result of 70% methanol green tea solution extract, Figure 2c is the result of 70% methanol extract aqueous fermentation tea. In FIG. 2A, OTA means okratoxin A.
Figure 3 is a graph showing the HPLC analysis (chromatogram) for the main components of tea according to an embodiment of the present invention. 3, G means gallic acid, EGC means (-)-epigallocatechin, C means catechin, CF means caffeine, and EGCG (- ) -epigallocatechin 3-Ο-gallate, ECG means (-)-epicatechin 3-Ο-gallate.
Figure 4 is purified by eluting 50% aqueous methanol solution using a Sep-Pak C18 cartridge (cartridge) of 70% methanol aqueous solution extract of green tea leaves according to an embodiment of the present invention as an elution solvent It is a graph showing the HPLC chromatogram for the (chromatogram). In FIG. 4, EGC means (-)-epigallocatechin, CF means caffeine, EGCG means (-)-epigallocatechin 3-Ο-gallate, and ECG means (-)-epicatechin 3- Means Ο-gallate.
5 is a tablet purified by eluting a 50% aqueous methanol solution using a Sep-Pak C18 cartridge (cartridge) of a 70% methanol aqueous solution extract of a microbial fermentation tea according to an embodiment of the present invention as an elution solvent It is a graph showing the HPLC chromatogram for water. In FIG. 5, G means gallic acid, EGC means (-)-epigallocatechin, C means catechin, CF means caffeine, and EGCG (- ) -epigallocatechin 3-Ο-gallate, ECG means (-)-epicatechin 3-Ο-gallate.

Hereinafter, preferred embodiments of the present invention are described. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

Example  One. HPLC Using Aflatoxin  And Okratoxin  Detection of A

Example  1-1. Preparation of sample

As a preliminary experiment to confirm the detection sensitivity of tea, especially microbial toxins Aflatoxin and Ochratoxin A, which are highly likely to remain in fermented tea, were first confirmed by HPLC.

As a microbial fermentation tea used in the experiment was used Chinese tea (Haehai tea window stilt bottle), green tea (Sullok tea, Pacific, South Korea) as a control. In addition, as a standard for checking sensitivity, aflatoxin is used in Transia ^? Standard reagent included in ELISA kits such as Plate Total Aflatoxin (BIOCONTROL, The Netherlands) was used, and olactoxin A was RIDASCREEN ^? Standard reagents included in ELISA kits of FAST Ochratoxin A (R-Biopharm, Germany) were used.

The microbial fermented tea samples and green tea samples were pretreated using methanol (DAE-JUNG, South Korea), that is, extracted with an aqueous methanol solution. Specifically, 20 g of each sample was added to 100 mL of an aqueous 70% methanol solution prepared by mixing methanol and tertiary distilled water in a volume ratio, followed by extraction with vigorous stirring on a shaker for 5 minutes, using a filter paper (Whatman No. 1). After filtration, the filtrate was used.

Example  1-2. HPLC Through Aflatoxin  analysis

After mixing 10 mL of the filtrate prepared in Example 1-1 and 10 mL of the second distilled water, 10 mL of the prepared mixture solution was eluted at a flow rate of 2 drop / sec on an immunoaffinity column without drying the immunoaffinity column, and then 10 mL of distilled water. Washed with and eluted with 3 mL acetonitrile. The eluted eluate was concentrated with nitrogen, deflated Aflatoxin using 100 μL of trifluoroacetic acid, and then dissolved by injecting 1.9 mL of HPLC mobile phase (20% acetonitrile) to dissolve 0.45. Passed through the μm filter was used as HPLC sample solution. HPLC analysis conditions for aflatoxin are shown in Table 1 below.

Item Conditions Instrument Shimadzu HPLC Column C18 4.6 x 50 mm Flow late 1.0 mL / min MobilePhase 25% acetonitrile Detector Fluorescence detector (Ex 360 nm, Em 418 nm)

In order to confirm the detection of aflatoxin, HPLC analysis was performed under the same conditions with respect to the standard, and the result of performing HPLC with the above method with respect to the standard and the sample is shown in FIG. 1.

As shown in Figure 1a, HPLC performed under the above conditions was confirmed to show a high sensitivity to the total aflatoxin (Total aflatoxin (B1, B2, G1, G2)). On the other hand, as shown in Figures 1b and 1c, the peak of the position corresponding to the aflatoxin of Figure 1a in both green tea and microbial fermented tea samples is not confirmed, the aflatoxin that is a fungal toxin is not detected in green tea and microbial fermented tea It was confirmed.

Example  1-3. HPLC Through Okratoxin  analysis

100 ml of 70% acetonitrile was added to each 25 g of the microorganism fermented tea sample or green tea sample of Example 1-1, and the extract obtained by homogenizing for 3 minutes in a blender was filtered by Whatman paper NO.1. Filtration gave a filtrate. After mixing 4 mL of the filtrate with 44 mL of the PBS solution, 10 mL of the prepared mixture was eluted at a flow rate of 2 drop / sec on an immunoaffinity column while the immunoaffinity column was not dried, washed with 10 mL of distilled water, and methanol 2 eluted with mL. The eluted eluate was concentrated with nitrogen, 500 μL of boron trifluoride methanol was added and reacted at 80 ° C. for 10 minutes to derivatize olactoxin A, followed by nitrogen concentration, and 1.9 mL of HPLC mobile phase (20% acetonitrile) was injected. After dissolving, the solution was passed through a 0.45 μm filter to obtain an HPLC sample solution. HPLC analysis conditions for Olatoxin A are shown in Table 2 below.

Item Conditions Instrument Shimadzu HPLC Column C18 4.6 x 50 mm Flow late 1.0 mL / min MobilePhase water: acetonitrile: acetic acid = 5: 5: 0.1 (v / v / v) Detector Fluorescence detector (Ex 333 nm, Em 460 nm)

In order to confirm the detection of okratoxin A, HPLC analysis was performed under the same conditions with respect to the standard, and the result of performing HPLC with the above method for the standard and the sample is shown in FIG. 2.

As shown in FIG. 2A, HPLC performed at these conditions was found to show high sensitivity to total okratoxin A. On the other hand, as shown in Figure 2b and 2c, the peak of the position corresponding to the okra toxin A of Figure 2a in both the green tea and microbial fermented tea sample was not confirmed, the fungal toxin okratoxin A in green tea and microbial fermented tea Was found not to be detected.

Example  2. ELISA kit Using Aflatoxin  And Okratoxin  Detection of A

Example  2-1. gun To aflatoxin  For detection experiment

The analysis of total aflatoxin was measured by selecting three manufacturers' products among commercial products. The assay kit used for the experiment was Transia ^? Plate Total Aflatoxin (BIOCONTROL, The Netherlands), RIDASCREEN ^? FAST Aflatoxin (R-Biopharm, Germany) and AgraQuant ^? Total Aflatoxin (ROMER, USA) was used, and the standard quantification range was 96 ppb to 80.0 ppb with 96 wells.

Assays were performed according to the manual of each kit. Although there was a slight difference in the order of each kit, the overall method was performed as follows. Specifically, 50 μL of the sample pretreated in Example 1-1 was added to each well of the ELISA kit, and then 50 μL of total Aflatoxin-HRP (Conjugate solution) and 50 μL of antibody (antibody solution) were sequentially added. Then, the ELISA kit to which the sample and each reaction solution was added was reacted at room temperature for 10 minutes in the dark, and after the reaction was completed, washed three times with distilled water to remove the reaction filtrate. Thereafter, 100 μL of the substrate solution was added and left at room temperature for 10 minutes, and then 100 μL of stop solution was added to terminate the reaction. After the reaction was completed, the ELISA kit was identified with a detection wavelength of 450 nm on an ELISA reader (BIORAD 680), and the total aflatoxin content was measured. The experiment was performed three times for each type of kit, and the average value of the measurement results is shown in Table 3 below. In Table 3, the numerical unit is ppb, and the background of 70% methanol used as a blank was 2 ppb to 4 ppb.

AgraQuant ^? Total aflatoxin RIDASCREEN ^? FAST Aflatoxin Transia ^? Plate Total Aflatoxin green tea 39.1 + - 14.0 341.3 ± 65.3 27.4 ± 4.7 Microbial Fermented Tea 40.2 ± 4.4 147.6 ± 43.9 21.5 ± 3.9

As shown in Table 3, aflatoxin above the reference level was detected in the green tea samples that did not undergo microbial fermentation in all three ELISA kits of different manufacturers. In addition, aflatoxin higher than the reference value was detected in the microbial fermented tea, and aflatoxin having a lower content than green tea was detected depending on the manufacturer.

The results may be inferred that the sample, that is, the green tea or microbial fermented tea used in the experiment was exposed to the strain producing mycotoxin, or contaminated with the produced fungal toxin, but aflatoxin as well as okratoxin A were almost It is arranged with the result of HPLC analysis of Example 1-2 which was not found, especially in the case of green tea, the fungus toxin in that the tea leaves are inactivated by heat treatment, and then vacuum-packed, and are not subjected to the microbial fermentation process. Since the possibility of contamination is very low, the result was estimated to be a result specifically generated by the components included in the tea during the fungal toxin analysis of the sample, that is, an error caused by the specific components included in the tea.

Example  2-2. Oklatoxin  A ( Ochratoxin  Detection experiment for A)

In the analysis of oklatoxin A, the sensitivity of the assay was measured by selecting two manufacturers' products among commercially available products. The assay kit used for the experiment was RIDASCREEN ^? FAST Ochratoxin A (R-Biopharm, Germany) or Ochratoxin-A EIA (EURO-DIAGNOSTICA, The Netherlands) was used, with standard wells ranging from 0.25 ppb to 50.0 ppb in 96 wells.

Assays were performed according to the manual of each kit. Although there was a slight difference in the order of each kit, the overall method was performed as follows. Specifically, 50 μL of the sample pretreated in Example 1-1 was added to each well of the ELISA kit, and then 50 μL of Ochratoxin-HRP (Conjugate solution) and 50 μL of the antibody solution were sequentially added. The ELISA kit to which the sample and the respective reaction solution were added was reacted at room temperature in the dark for 10 minutes, and after the reaction was completed, the reaction filtrate was washed three times with distilled water to remove the reaction filtrate. Thereafter, 100 μL of the substrate solution was added and left at room temperature for 10 minutes, and then 100 μL of stop solution was added to terminate the reaction. After the reaction was completed, the ELISA kit was confirmed to have a detection wavelength of 450 nm on an ELISA reader (BIORAD 680) to measure the okratoxin content. The experiment was performed three times for each type of kit, and the average value of the measurement results is shown in Table 4 below. In Table 4, the numerical unit is ppb, and the background of 70% methanol used as a blank was 2 ppb to 4 ppb.

RIDASCREEN ^? AST Ochratoxin A Ochratoxin-A EIA green tea 60.0 ± 2.3 97.7 ± 16.1 Microbial Fermented Tea 40.2 ± 1.1 68.2 ± 9.2

As shown in Table 4, okratoxin A above the reference level was detected in the green tea samples that do not undergo microbial fermentation in both EL EL kits of different manufacturers. In addition, in the microbial fermented tea, okratoxin A above the reference value was detected, but aflatoxin having a lower content than green tea was detected.

The results may be inferred that the sample, that is, the green tea or microbial fermented tea used in the experiment was exposed to the strain producing mycotoxin, or contaminated with the produced fungal toxin, but aquatoxin A as well as aquatoxin A It is arranged with the result of HPLC analysis of Example 1-2 and Example 1-3 which were not found, and especially in the case of green tea, the tea leaves are inactivated by heat treatment, and then vacuum-packed and stored. Since it is very unlikely to be contaminated with fungal toxins, the results indicate that the results are caused specifically by the components contained in the tea during the fungal toxin analysis of the sample. It was estimated.

Example  3. ELISA Kit  Determine optimal pretreatment conditions for the analysis

In order to determine the optimum pretreatment conditions for analyzing aflatoxin and olatoxin A using an ELISA kit from 70% methanol extract samples of green tea and microbial fermented tea prepared in Example 1, first, Sep-Pak ^? Extract samples were purified using a C18 cartridge 6cc (Waters, USA). More specifically, after activating the C18 cartridge using distilled water and methanol, 3 mL of each of the methanol extract samples prepared in Example 1 was put into the cartridge and concentrated under nitrogen. Then, 100% distilled water, 20% aqueous methanol solution, 50% aqueous methanol solution, 70% aqueous methanol solution and 100% methanol were sequentially injected and 3 mL, respectively, eluted, and the extract was divided into five fractions. Components were analyzed using HPLC and ELISA kits. In addition, using the same method as the total aflatoxin standard reagent and okratoxin A standard reagent used in Example 2, respectively, 80 ppb was confirmed whether the detection in the ELISA kit for each fraction layer. The assay using the ELISA kit was performed in the same manner as in Example 2.

HPLC analysis conditions for the HPLC analysis are shown in Table 5.

Items Condition Instrument Jasco HPLC Column Cosmosil C18 4.6 x 50mm Flow rate 0.9 mL / min Oven temp. 40 Detector UV 275 nm Mobile phase time (min) acetonitrile (%) 50 Mm H ₃ PO ₄ (%) (Gradient) 0 8 92 55 31 69 65 50 50

First, an analysis method using an ELISA kit was performed on the fraction layer of total aflatoxin standard reagent and okratoxin A standard reagent in the same manner as in Example 2. With regard to the total aflatoxin, Transia ^? Plate Total Aflatoxin was used, and RIDASCREEN ^? FAST Ochratoxin A was used. The analysis results are shown in Table 6 below.

C18 cartridge
Elute unit ; ppb Total aflatoxin Ochratoxin a 100% H ₂ O 6 3 20% methanol 7 6 50% methanol 10 11 70% methanol 64 71 100% methanol 9 6

As shown in Table 6, when the fungal toxin fractionated with Sepak-C18 column, 64 ppb of total aflatoxin was detected in the fraction layer eluted with 70% aqueous methanol solution, and the recovery rate was about 80%. In the case of 71 ppb was detected showing a recovery of about 88.8%. From the above results, it was confirmed that both total aflatoxin and olatoxin A, which were fungal toxins, had the highest detection sensitivity in the fraction layer eluted with 70% aqueous methanol solution.

In addition, an analysis method using an ELISA kit was performed on the fraction layer of the green tea sample and the microbial fermented tea sample prepared in Example 1 in the same manner as in Example 2. Regarding the total aflatoxin, as described above, Transia ^? Plate Total Aflatoxin was used, and RIDASCREEN ^? FAST Ochratoxin A was used. The analysis results are shown in Table 7 below.

C18
cartridge
Elute unit ; ppb green tea Microbial Fermented Tea Total aflatoxin Ochratoxin a Total aflatoxin Ochratoxin a 100% H ₂ O 8 3 7 One 20% methanol 3 8 9 7 50% methanol 45 52 33 35 70% methanol 4 5 4 6 100% methanol 4 4 3 4

As shown in Table 7, green tea showed high values of both aflatoxin and okratoxin A in the fraction layer eluted with 50% aqueous methanol solution. In addition, in the case of microbial fermented tea, both aflatoxin and olatoxin in the fraction layer eluted with 50% methanol aqueous solution showed high values.

Compared with the results of Table 6, the components contained in the fraction layer eluted with 50% aqueous methanol solution was confirmed that the other components other than aflatoxin or olatoxin A, that is, the interference factor of detection by the ELISA kit, the 50 Analysis of the components contained in the fraction layer eluted with% methanol aqueous solution was performed using HPLC.

In this regard, first, HPLC for the main components of tea was carried out by the method of Table 5 above. More specifically, gallic acid (epicatechin), epigallocatechin ((-)-epigallocatechin), epigallocatechin ((-)-epigallocatechin 3 purchased from Sigma Co., USA -Ο-gallate), epicatechin gallate ((-)-epicatechin 3-Ο-gallate), catechin ((+)-catechin) and caffeine standard reagents were purchased from Sigma Co. HPLC was performed under the conditions of Table 5 above. The results of the above are shown in FIG.

In addition, HPLC was performed on the fraction layer eluted with 50% methanol solution of green tea and the fraction layer eluted with 50% methanol solution of microbial fermentation tea, respectively. 5 is shown.

As shown in FIG. 4, in the case of the green tea extract sample, compared with FIG. 3, (-)-epicatechin 3-Ο-gallate (ECG), caffeine (C), and (-)-epigallocatechin 3-Ο-gallate (EGCG) and (-)-epigallocatechin (EGC) were detected as main components, and the detected concentrations were 3,700 ppm for ECG, 3,500 ppm for C, 5,400 ppm for EGCG, and 3,700 ppm for EGC.

In addition, as shown in FIG. 5, in the case of the microbial fermented tea sample, gallic acid (G) and caffeine (C) were mainly detected as the main detection components, and the detected concentrations were 3,500 ppm, respectively. And 3,500 ppm.

HPLC analysis of 50% methanol elution fractionation layer in Sepak-C18 cartridge among 70% methanol extract samples of green tea and microbial fermented tea showed that the main detection component could be an obstacle to tea sample analysis using ELISA kit. Presumed to be a substance, an assay using an ELISA kit was performed for each standard in the same manner as in Example 2. Measurement concentration of the main detection component With respect to the total aflatoxin, as described above, Transia ^? Plate Total Aflatoxin was used, and RIDASCREEN ^? FAST Ochratoxin A was used. The analysis results are shown in Table 8 below.

Standard reagent Concentration (ppm) Total aflatoxin (ppb) Ochratoxin (ppb) G 3,500 9 50 EGC 3,700 20 23 C 3,500 42 13 EGCG 5,400 15 44 ECG 3,700 15 38

As shown in Table 8, the major components of tea contained in the eluate of 50% methanol aqueous solution of green tea samples and microbial fermented tea samples were tested with a standard reagent, and all five species were ELISA for total aflatoxin and okratoxin A. It was confirmed that it acts as an obstacle in the detection reaction using the kit. In particular, caffeine (C) was found to have a significantly stronger interference with total aflatoxin in total aflatoxin ELISA kits, and gallic acid (G) was identified as the largest blocker in olatoxin A ELISA kits.

Example  4. ELISA kit Of pretreatment effect to improve reliability

From the results of Example 3, (-)-epigallocatechin 3-Ο-gallate (EGCG), (-)-epicatechin 3-Ο-gallate (ECG), (-) -epigallocatechin (EGC), gallic acid (G) and caffeine (C) were all identified as major interferers of the total aflatoxin ELISA kit and the okratoxin A ELISA kit. On the other hand, it was confirmed that the components are mostly eluted in the fractionation layer of the 50% methanol aqueous solution eluting solvent during the purification process using a C18 cartridge.

Therefore, in case of using a fractional layer in which 70% methanol solution eluted with total aflatoxin and olatoxin A is eluted as an elution solvent, that is, 50% methanol solution is eluted using a Sep-Pak C18 cartridge for methanol extract of microbial fermented tea. Removing the eluate using a solvent, obtaining an eluate using a 70% methanol solution elution solvent, when analyzing the obtained eluate using an ELISA kit, the interference factor is sufficiently removed to obtain a reliable result It was expected to be.

In the 70% methanol solution of the microbial fermented tea sample, the total aflatoxin and okratoxin A were each spiked at a concentration of 80 ppb, and then charged into a Sep-Pak C18 cartridge, and the method of Example 3 As a result, a fraction layer eluted with each eluting solvent was obtained, and the fraction layer was analyzed using an ELISA kit in the same manner as in Example 3. The analysis results are shown in Table 9.

C18 cartridge Elute Total Aflatoxin (ppb) Ochratoxin A (ppb) 100% H ₂ O 9.3 ± 0.6 2.3 ± 0.6 20% methanol 11.7 ± 2.1 18.3 ± 0.6 50% methanol 40.0 ± 2.0 39.0 ± 2.0 70% methanol 67.7 ± 2.5 70.0 ± 3.1 100% methanol 6.0 ± 1.0 7.0 ± 1.0

As shown in Table 9, the highest total aflatoxin and olatoxin A were identified in the fractionation layer eluted with 70% aqueous methanol solution as an eluting solvent. , 67.7 ppb was recovered and except for 4 ppb confirmed in Table 7, a recovery rate of about 79.6% was confirmed, in the case of okratoxin A, 70.0 ppb recovered to exclude 6 ppb identified in Table 7 In this case, a recovery rate of about 82.5% was confirmed. On the other hand, in the case of the fraction layer eluted with 50% methanol solution as the eluting solvent, except for the 33 ppb and 35 ppb identified in Table 7, only about 7 ppb or 4 ppb is confirmed, the level slightly out of the error range Confirmed.

The results in Table 9 are artificially added to the microbial toxin, but the microbial fermentation tea used in the experiment was added only because it contains almost no microbial toxin, so from the above results, Sep-Pak for methanol extract of the microbial fermentation tea While performing the C18 cartridge fractionation method (SPE extraction method or SPE separation method), the fractionation layer in which 50% aqueous methanol solution was eluted with elution solvent was removed, and only the fractionation layer in which 70% aqueous methanol solution was eluted with elution solvent was obtained. When using the enzyme immunoassay, it is economical because it is cheaper than HPLC, simple operation is possible, and no skilled technician is required, and the process can be performed in a short time compared to HPLC. In addition, various types of hazards such as green tea, black tea, and oolong tea that can grow microorganisms during storage Microbial toxins, in particular, are evaluated in a way that can quickly and effectively detect total aflatoxins and Ochratoxin A for.

Claims (6)

Preparing a solvent extract using tea leaves using at least one selected from the group consisting of water and alcohols having 1 to 5 carbon atoms;
Preparing a purified product by eluting a 70% methanol aqueous solution with an elution solvent using a Sep-Pak C18 cartridge; And
Measuring the fungal toxin (Mycotoxin) from the purified by enzyme immunoassay
Method of analyzing fungal toxins from tea comprising. The method of claim 1,
The fungal toxin is a method for analyzing fungal toxin from at least one selected from the group consisting of Aflatoxin (Aflatoxin) and Ochratoxin A (Ochratoxin A). The method of claim 1,
The tea is a method for analyzing fungal toxins from any one selected from the group consisting of microbial fermented tea, green tea, green tea, black tea, oolong tea and mixtures thereof. The method of claim 1,
The solvent of the step of preparing a solvent extract is a method for analyzing fungal toxins from tea, characterized in that any one selected from the group consisting of water, methanol and mixtures thereof. The method of claim 1,
The enzyme immunoassay method is to analyze the fungal toxin from tea that is performed using an antibody against any one of the fungal toxin selected from the group consisting of Aflatoxin (Aflatoxin) and Okraatoxin A (Ochratoxin A). The method of claim 5,
The enzyme immunoassay is characterized in that an ELISA (Enzyme-linked immunosorbent assay) is performed by using an antibody against any one fungal toxin selected from the group consisting of Aflatoxin and Ochratoxin A. To analyze fungal toxins from bacteria.

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