KR101287340B1 - A microplate method for quantifying red pigments of pepper fruit - Google Patents

A microplate method for quantifying red pigments of pepper fruit Download PDF

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KR101287340B1
KR101287340B1 KR1020110113163A KR20110113163A KR101287340B1 KR 101287340 B1 KR101287340 B1 KR 101287340B1 KR 1020110113163 A KR1020110113163 A KR 1020110113163A KR 20110113163 A KR20110113163 A KR 20110113163A KR 101287340 B1 KR101287340 B1 KR 101287340B1
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한정헌
홍순철
이준대
윤재복
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(주)고추와 육종
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Abstract

본 발명은 고추 과실의 붉은 색소 함량 분석을 위한 마이크로플레이트(microplate)법에 관한 것으로, 더욱 상세하게는 폴리스티렌 재질의 마이크로플레이트(microplate)를 고춧가루의 붉은 색소 분석에 적용할 수 있도록, 붉은 색소 추출 용매인 아세톤에 폴리스티렌 재질이 녹지 않게 폴리스티렌에 대한 아세톤의 반응력을 식용유를 이용하여 저하시켜 96개 시료를 동시에 분석할 수 있는 방법에 관한 것이다. 이를 위하여 본 발명은 2ml 원심분리용 튜브에 아세톤 1ml와 고춧가루 0.1g를 넣고 붉은 색소를 추출하는 단계, 8시간 동안 추출 후 원심분리기로 13,000rpm으로 1분 동안 원심분리하는 단계, 상층액 100ul와 식용유 900ul를 새로운 1.5ml 원심분리용 튜브에 넣고 혼합하는 단계, 혼합액 200ul를 마이크로플레이트(microplate)에 분주하고 효소면역측정장비(ELISA reader)를 이용하여 460nm 혹은 450nm 파장에서 흡광도를 측정하는 단계, 마이크로플레이트(microplate)법으로 얻은 흡광값에 상수 0.8611을 곱하고 0.07을 더해주어 아스타(ASTA)법에 의한 색소 함량 결과를 추정하는 단계로 이루어진 것에 특징이 있다.The present invention relates to a microplate method for analyzing the red pigment content of red pepper fruit, and more particularly, to apply a microplate made of polystyrene to the red pigment analysis of red pepper powder, a red pigment extraction solvent. It relates to a method of analyzing 96 samples at the same time by reducing the reaction force of acetone to polystyrene using cooking oil so that the polystyrene material does not melt in the acetone. To this end, in the present invention, 1 ml of acetone and 0.1 g of red pepper powder are added to a 2 ml centrifuge tube to extract red pigment, and after 8 hours of extraction, centrifugation at 13,000 rpm for 1 minute with a supernatant 100ul and cooking oil. Put 900ul into a new 1.5ml centrifuge tube and mix, dispense 200ul of the mixed solution into a microplate and measure the absorbance at 460nm or 450nm wavelength using an ELISA reader, microplate The absorbance value obtained by the (microplate) method is multiplied by a constant of 0.8611 and added to 0.07 is characterized by consisting of the step of estimating the pigment content results by the Asta (ASTA) method.

Description

고추 과실의 붉은 색소 함량 분석을 위한 마이크로플레이트법{A microplate method for quantifying red pigments of pepper fruit}A microplate method for quantifying red pigments of pepper fruit

본 발명은 고춧가루 붉은 색소를 신속하게 대량으로 함량 분석할 수 있는 환경 친화적인 방법 개발에 관한 것이다. 상세하게는 폴리스티렌 재질의 마이크로플레이트를 고춧가루 붉은 색소 분석에 적용할 수 있도록, 붉은 색소 추출 용매인 아세톤에 폴리스티렌 재질이 녹지 않게 폴리스티렌에 대한 아세톤의 반응력을 식용유를 이용하여 저하시켜 96개 시료를 동시에 분석할 수 있는 방법이다. 또한 색소분석에 주로 이용되고 있는 미국양념무역협회(American Spice Trade Associantion, ASTA)법에 비해 색소추출에 요구되는 아세톤의 양(100ml)을 최소 1/100 이하로 줄여 환경오염을 최소화한 분석법이다.The present invention relates to the development of an environmentally friendly method capable of quickly and mass content analysis of red pepper powder red pigment. Specifically, in order to apply a polystyrene-based microplate to red pepper analysis, redox extraction solvent reduced acetone's reaction force to polystyrene by using cooking oil so that the polystyrene material did not dissolve in acetone. This is how you can do it. In addition, it is an analysis method that minimizes environmental pollution by reducing the amount of acetone (100 ml) required for pigment extraction to at least 1/100 of the amount compared to the American Spice Trade Associantion (ASTA) method, which is mainly used for pigment analysis.

고추의 붉은 색소는 식품(고춧가루나 고추장)의 품질에 영향을 주는 주요 인자인 동시에 화장품의 천연색소 원료로도 사용된다. 신미와 색소 추출재료로 세계 각국에서 생산된 올레오레진(oleoresin)의 총생산량은 2006년을 기준으로 약 540톤 정도이며, 국내에서는 2000년 이후 올레오레진(oleoresin)을 지속적으로 수입하고 있다. 이처럼 고추의 붉은 색소는 산업적으로 매우 중요하기 때문에 육종에 있어서는 고색소 품종 개발에 대한 요구가 늘어나고 있는 추세이다. The red pigment of red pepper is a major factor affecting the quality of food (pepper powder or red pepper paste) and is also used as a natural pigment source of cosmetics. The total output of oleoresin produced from all over the world as an extract of pigment and pigment is around 540 tons as of 2006, and Korea has been continuously importing oleoresin since 2000. As such, the red pigment of red pepper is very important industrially, the demand for the development of high pigment varieties is increasing in breeding.

고색소 고추 품종을 육성을 위해서는 고추의 색소를 효과적으로 분석할 수 있는 방법이 필요한데, 색도계(colorimeter) 또는 분광광도계를 이용하는 방법, 정밀한 측정을 위해 고성능 액체크로마토그래피분석법(high-performance liquid chromatography, HPLC), 액체크로마토그래피/질량분광분석법(liquid chromatography/mass spectrometry, LC/MS), 자기공명분석법(nuclear magnetic resonance, NMR) 등을 이용하는 방법이 있다. 일반적으로 붉은 색소 함량 분석에는 미국양념무역협회(American Spice Trade Associantion, ASTA)에서 규정한 아스타(ASTA) 측정법이 주로 이용되고 있는데, 신속하고 간편하게 다량의 시료를 분석하는 데에는 많은 어려움이 있다.   In order to cultivate high-pigmented pepper varieties, a method of effectively analyzing the pigments of peppers is required, using a colorimeter or spectrophotometer, and high-performance liquid chromatography (HPLC) for precise measurement. , Liquid chromatography / mass spectrometry (LC / MS), nuclear magnetic resonance (NMR), and the like. In general, the analysis of red pigment content is mainly used by the American Spice Trade Associantion (ASTA) measuring method (ASTA), which is difficult to analyze a large amount of samples quickly and easily.

아스타(ASTA) 측정법에 의한 붉은 색소 측정 과정을 살펴보면, 먼저 도 1에서와 같이 고춧가루 0.1g과 아세톤 100ml을 200ml 플라스틱병(1)에 담고, 교반기나 회전교반기를 이용하여 실온에서 8시간 동안 반응시킨 다음 고춧가루를 제외한 상층액을 흡광값 측정에 사용한다. 색소를 포함하는 아세톤액을 5ml 유리 큐벳(cuvette)(3)에 3ml 첨가하고 흡광분석기(spectrophotometer)를 이용하여 460nm에서 흡광값을 측정하고, 흡광값에 16.4를 곱한 값을 색소함량으로 사용한다.   Looking at the red pigment measurement process by the Asta (ASTA) method, first, as shown in Figure 1, 0.1g red pepper powder and acetone 100ml in a 200ml plastic bottle (1), and reacted for 8 hours at room temperature using a stirrer or a stirrer The supernatant except for the following red pepper powder is used to measure the absorbance value. 3 ml of acetone solution containing a dye was added to a 5 ml glass cuvette (3), and the absorbance value was measured at 460 nm using a spectrophotometer, and the absorbance value multiplied by 16.4 was used as the pigment content.

그러나 이와 같은 방법에 의해 붉은 색소를 측정할 경우, 색소 추출에 다량의 유기용매(주로 아세톤)를 사용한다는 점, 환기시설 있는 곳에서 다량의 시료를 한 번에 조제하기가 매우 힘들다는 점, 색소 측정 용기 세척을 위해 부가적인 아세톤을 소비해야 한다는 점 등 번거로운 작업 절차와 환경오염에 관한 다수의 문제점이 있다는 것이다.   However, when red pigments are measured by this method, a large amount of organic solvent (mainly acetone) is used to extract the pigments, and it is very difficult to prepare a large amount of samples at once in a ventilated facility. There are a number of problems with cumbersome work procedures and environmental pollution, such as the need to consume additional acetone for cleaning the measuring vessel.

해당사항 없음None

[문헌1] The Official Analytical Methods of the American Spice Trade Association. An electronic version of 20.1. Extractable Color in Capsicums and Their Oleoresins (Revised October 2004) (http://www.astaspice.org/i4a/pages/index.cfm?pageid=3745)1 The Official Analytical Methods of the American Spice Trade Association. An electronic version of 20.1. Extractable Color in Capsicums and Their Oleoresins (Revised October 2004) (http://www.astaspice.org/i4a/pages/index.cfm?pageid=3745)

본 발명에서는 다량의 고춧가루 시료에서 붉은 색소 함량을 신속하게 친환경적으로 분석할 수 있는 방법을 개발하는데 그 목적이 있다. 이를 위해 도 1에서처럼 효소면역항체법 등 생물과 관련된 다양한 실험에 이용되는 폴리스티렌 재질의 마이크로플레이트(microplate)를 색소 측정에 활용하는데 필요한 주요 문제점과 제반 조건을 조사하였다. In the present invention, an object of the present invention is to develop a method for rapidly analyzing environmentally friendly red pigment content in a large amount of red pepper powder samples. To this end, the main problems and conditions required for the use of microstyrene (microplate) made of polystyrene used in various experiments related to organisms such as enzyme-immune antibody method for the measurement of pigments were investigated.

본 발명은 고춧가루의 붉은 색소 함량을 측정하는데 있어서, 2ml 용량의 원심분리용 튜브에 고춧가루 0.1g을 정량하고, 아세톤 1ml을 첨가한 다음 상온에서 8시간 반응시켜 색소를 추출하는 단계, 상기 붉은 색소를 함유한 아세톤 100ul를 900ul 식용유와 고르게 혼합시켜줌으로써 폴리스티렌과 아세톤의 반응력을 낮추는 단계, 아세톤과 식용유 혼합액을 96-웰(well) 마이크로플레이트(microplate)에 200ul 첨가 후 460nm 또는 450nm에서 흡광도를 측정하는 단계, 시료의 흡광도 값을 환산하여 아스타(ASTA)법에 의해 측정된 수치로 환산하는 단계로 이루어진 것에 특징이 있다.In the present invention, red pigment content of red pepper powder is measured, and 0.1g of red pepper powder is quantified in a centrifuge tube having a volume of 2 ml, 1 ml of acetone is added and then reacted at room temperature for 8 hours to extract the pigment, the red pigment Lowering the reaction force of polystyrene and acetone by mixing 100ul of acetone with 900ul cooking oil evenly, and measuring the absorbance at 460nm or 450nm after adding 200ul of acetone and cooking oil mixture to a 96-well microplate. , Converting the absorbance value of the sample into a value measured by the Asta (ASTA) method.

본 발명에서는 96개 시료를 동시에 분석할 수 있는 흡광값 측정 장비인 효소면역측정장비(ELISA reader)를 붉은 색소 함량 분석에 이용하고자 하였다. 이를 위해 흡광값 측정에 사용되는 폴리스티렌 재질의 96-웰(well) 마이크로플레이트(microplate) 표면이 붉은 색소 추출용매인 아세톤에 의해 변화되는 것을 식용유를 이용하여 차단하였다. 결과적으로 다량의 고춧가루 시료에서 색소 함량을 분석할 수 있음은 물론이고, 색소 추출용기를 소형화함으로써 다량의 고춧가루 시료로부터 색소추출이 용이해졌으며, 기존의 아스타(ASTA) 방법에 비해 1/100 이하 수준으로 사용하는 아세톤의 양을 줄임으로써 환경에 해로운 폐액 발생량을 현저히 감소시켰다.In the present invention, an enzyme immunoassay device (ELISA reader), which is an absorbance measurement device capable of simultaneously analyzing 96 samples, was used for red pigment content analysis. For this purpose, the surface of the 96-well microplate made of polystyrene used for measuring the absorbance value was blocked by cooking oil using acetone, a red pigment extraction solvent. As a result, it is possible not only to analyze the pigment content in a large amount of red pepper powder sample, but also by miniaturizing the pigment extraction container, it is easy to extract the color from a large amount of red pepper powder sample, and it is 1/100 or less than the conventional ASTA method. By reducing the amount of acetone used, the amount of waste fluid harmful to the environment was significantly reduced.

도 1은 아스타(ASTA)법과 마이크로플레이트(microplate)법을 이용한 고춧가루 붉은 색소의 분석과정을 나타낸 모식도.
도 2는 고춧가루 붉은 색소의 파장별 흡광값 비교도.
도 3은 본 발명에 따른 붉은 색소 함량 분석을 위한 아스타(ASTA)법과 마이크로플레이트(microplate)법의 상관관계 비교도.
도 4는 본 발명에 따른 마이크로플레이트(microplate)법에 의한 붉은 색소 함량 분석 예.Figure 1 is a schematic diagram showing the analysis process of red pepper powder red pigment using the Asta (ASTA) method and microplate (microplate) method.
Figure 2 is a comparison of the absorbance value for each wavelength of red pepper powder.
Figure 3 is a comparison of the correlation between the ASTA (ASTA) method and the microplate (microplate) method for red pigment content analysis according to the present invention.
Figure 4 is an example red pigment content analysis by the microplate (microplate) method according to the present invention.

본 발명의 구체적인 방법을 첨부된 도면을 참조하여 상세히 설명하면 다음과 같다.   The detailed method of the present invention will be described in detail with reference to the accompanying drawings.

도 1은 고춧가루의 붉은 색소 함량 측정을 위해 사용되는 아스타(ASTA)법과 본 발명에서 고안한 마이크로플레이트(microplate)법을 이용한 고춧가루 붉은 색소 분석과정을 비교한 도면이다.
기존 아스타(ASTA)법에서는 200ml 플라스틱병(1)에 고춧가루 0.1g과 아세톤 100ml를 넣고 회전교반기를 이용하여 실온에서 8시간 동안 반응시켜 색소를 추출한다. 반응액을 2-3분간 정치시킨 다음 고춧가루를 제외한 상층액 3ml를 5ml 유리 큐벳(cuvette)(3)에 첨가하고 흡광분석기를 이용하여 460nm에서 흡광도를 측정한다.그러나 본 발명의 마이크로플레이트법에서는 2ml 용량의 상업용 원심분리용 튜브(2)에 고춧가루 0.1g과 아세톤 1ml를 넣고 회전교반기를 이용하여 실온에서 8시간 동안 반응시켜 색소를 추출한다. 반응액은 원심분리기로 13,000rpm으로 1분 동안 원심분리한 다음 고춧가루를 제외한 상층액 100ul를 900ul의 식용유가 미리 담긴 1.5ml 원심분리용 튜브에 넣고 두 용액이 잘 섞일 수 있도록 교반기(vortexer)를 이용하여 10초간 혼합한다. 아세톤이 식용유에 완전히 혼합되면 폴리스티렌과 반응하는 아세톤의 기능기가 식용유에 의해 차단되어 흡광도 측정에 이용되는 폴리스티렌 재질의 96-웰(wells) 마이크로플레이트(microplate) 표면을 변형시키지 않는다. 결과적으로 색소를 포함하는 아세톤을 마이크로플레이트(microplate)에 담을 수 있고, 아세톤과 식용유 모두 색소와 반응하는 기능기에는 영향을 받지 않기 때문에 색소 함량 분석을 위한 이후의 단계를 더 진행할 수 있게 된다. 최종적으로 폴리스티렌 재질의 96-웰(wells) 마이크로플레이트(microplate)(5)에 200ul의 혼합액을 시료별로 각각의 웰(well)에 분주하고 효소면역측정장비(ELISA reader)를 이용하여 450nm에서 혼합액의 흡광도를 측정한다. 1 is a view comparing the red pepper powder analysis process using the ASTA method used to measure the red pigment content of red pepper powder and the microplate method developed in the present invention.
In the conventional ASTA method, red pepper powder 0.1g and acetone 100ml are put into a 200ml plastic bottle (1) and reacted for 8 hours at room temperature using a rotary stirrer to extract a pigment. The reaction solution was allowed to stand for 2-3 minutes, and then 3 ml of the supernatant except for red pepper powder was added to a 5 ml glass cuvette (3), and the absorbance was measured at 460 nm using an absorbance analyzer. However, in the microplate method of the present invention, 2 ml was used. 0.1 g of red pepper powder and 1 ml of acetone were added to a commercial centrifuge tube 2 having a capacity, and the resultant was reacted for 8 hours at room temperature using a rotary stirrer to extract a pigment. The reaction solution was centrifuged at 13,000 rpm for 1 minute using a centrifuge, and 100 ul of the supernatant except for red pepper powder was placed in a 1.5 ml centrifuge tube containing 900 ul of cooking oil in advance, using a vortexer to mix the two solutions well. Mix for 10 seconds. When acetone is thoroughly mixed with cooking oil, functional groups of acetone reacting with polystyrene are blocked by the cooking oil so as not to modify the surface of the polystyrene 96-well microplate used for absorbance measurement. As a result, the acetone containing the dye can be contained in a microplate, and since acetone and cooking oil are not affected by the functional groups reacting with the dye, further steps for the dye content analysis can be performed. Finally, 200 μl of the mixed solution was dispensed into each well for each sample in a 96-well microplate 5 made of polystyrene, and the mixture was mixed at 450 nm using an ELISA reader. Measure the absorbance.

도 2는 원하는 파장을 선택할 수 있는 효소면역측정장비(ELISA reader)뿐만 아니라 특정 파장만 측정 가능한 효소면역측정장비(ELISA reader)로도 색소를 측정할 수 있도록 파장에 따른 적색소의 흡광값 변화를 조사한 것이다. 흡광측정기(spectrophotometer)로 특정 파장만 측정 가능한 효소면역측정장비(ELISA reader)에서 일반적으로 측정 가능한 450nm와 405nm의 파장과 아스타(ASTA)법에서 권장하는 460nm에서 각 파장별로 11개 시료의 흡광값을 시료 당 3회 측정한 결과, 450nm와 460nm의 흡광값에는 오차범위에서 차이가 없음을 확인할 수 있었다.   Figure 2 is to investigate the change in the absorbance value of the red pigment according to the wavelength so that the dye can be measured by the enzyme immunoassay device (ELISA reader) that can select the desired wavelength, as well as the ELISA reader that can measure only a specific wavelength. . Absorbance values of 11 samples for each wavelength at wavelengths of 450 nm and 405 nm, typically measured by ELISA readers that can only measure specific wavelengths with a spectrophotometer, and at 460 nm, as recommended by the Asa method. As a result of three measurements per sample, it was confirmed that there was no difference in the absorbance values between 450 nm and 460 nm.

도 3은 도 2의 자료를 근거로 450nm에서 색소 함량을 측정할 때, 아스타(ASTA)법과 본 발명에 따른 마이크로플레이트(microplate)법 사이에 어느 정도의 상관성이 있는지를 조사한 것인데, 두 방법 간에는 98%이상 유의수준이 있는 것으로 확인되어 본 발명에 따른 마이크로플레이트(microplate)법에 의한 고춧가루 붉은 색소 함량 측정이 매우 정확한 방법임을 확인할 수 있었다. 마이크로플레이트(microplate)법으로 얻은 흡광값에 상수 0.8611을 곱하고 0.07을 더해주면 아스타(ASTA)법의 색소 함량 결과와 98% 이상 유의성을 갖는다.   Figure 3 is based on the data of Figure 2 when measuring the pigment content at 450nm, to investigate the degree of correlation between the ASTA method and the microplate method according to the present invention, the 98 between the two methods It was confirmed that there is a significance level of more than%, it was confirmed that the red pigment content measurement of red pepper powder by the microplate method according to the present invention is a very accurate method. When the absorbance value obtained by the microplate method is multiplied by a constant of 0.8611 and added to 0.07, the result of the pigment content of the ASTA method is more than 98% significant.

(실시예) 도 4는 본 발명에 따른 마이크로플레이트(microplate)법을 여러 가지 고추 시료의 붉은 색소 함량에 측정에 이용한 예이다.(Example) Fig. 4 is an example of using the microplate method according to the present invention to measure the red pigment content of various pepper samples.

본 발명에 따른 마이크로플레이트(microplate)법은 고추 가루의 붉은 색소 함량 측정뿐만 아니라 아세톤으로 추출 가능하고 특정 파장에서 고유의 흡광값을 가지는 색소의 함량 측정에 응용될 수 있다.The microplate method according to the present invention can be applied not only to the measurement of the red pigment content of red pepper powder, but also to the content of the pigment which can be extracted with acetone and has a specific absorbance value at a specific wavelength.

해당사항 없음None

Claims (2)

2ml 원심분리용 튜브에 아세톤 1ml와 고춧가루 0.1g를 넣고 붉은 색소를 추출하는 단계, 8시간 동안 추출 후 원심분리기로 13,000rpm으로 1분 동안 원심분리하는 단계, 상층액 100ul와 식용유 900ul를 새로운 1.5ml 원심분리용 튜브에 넣고 혼합하는 단계, 혼합액 200ul를 마이크로플레이트(microplate)에 분주하고 효소면역측정장비(ELISA reader)를 이용하여 460nm 혹은 450nm 파장에서 흡광도를 측정하는 단계로 이루어진 일련의 방법(마이크로플레이트분석법)을 이용하여 고춧가루의 붉은 색소 함량을 분석하는 방법1 ml of acetone and 0.1 g of red pepper powder in a 2 ml centrifuge tube, extracting red pigment, extracting for 8 hours, centrifuging at 13,000 rpm for 1 minute with a centrifuge, and adding 1.5 ul of supernatant 100 ul and 900 ul of cooking oil. A series of methods consisting of a step of mixing in a tube for centrifugation, dispensing 200 ul of the mixed solution into a microplate and measuring absorbance at a wavelength of 460 nm or 450 nm using an ELISA reader (microplate). Method for analyzing red pigment content of red pepper powder 청구항 제1항에 따른 마이크로플레이트분석법을 이용하여 아세톤 추출이 가능한 색소의 함량을 분석하는 방법Claim 1 method for analyzing the content of acetone extractable pigment using the microplate analysis method according to claim 1
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KR20050069252A (en) * 2003-12-31 2005-07-05 주식회사 두산 Method for determining the extractable color in capsicums using sonication

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KR20050069252A (en) * 2003-12-31 2005-07-05 주식회사 두산 Method for determining the extractable color in capsicums using sonication

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