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들깨 잎의 FT-IR 스펙트럼 데이터로부터 다변량 통계분석을 이용한 생산연도 판별

서혜영1, 서은지1, 최은빈1, 이미자1, 이한결1, 서우덕1, 김정인2, 송승엽1,*

Determination of Production Year Using Multivariate Statistical Analysis from FTIR Spectrum Data of Perilla Leaves

Korean Journal of Breeding Science 2024;56(1):11-18.
Published online: March 1, 2024

1농촌진흥청 국립식량과학원 작물기초기반과

2농촌진흥청 국립식량과학원 남부작물부 밭작물개발과

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*Corresponding to Seung-Yeob SongTEL. +82-63-238-5336E-mail. s2y337@korea.kr
• Received: November 24, 2023   • Revised: December 12, 2023   • Accepted: December 14, 2023

Copyright © 2024 by the Korean Society of Breeding Science

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • This study used perilla seeds produced in 2019, 2020, and 2021 to determine the year of production using multivariate statistical analysis of Fourier-transform infrared (FTIR) spectral data of perilla leaves. Spectral analysis based on multivariate statistical analysis of whole-cell extracts was used to distinguish the perilla leaves at the metabolic level. FT-IR spectral data of the leaves were analyzed using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The FTIR spectrum identified spectral differences between the frequency regions of 1,700 to 1,500, 1,500 to 1,300, and 1,100 to 950 cm-1. This spectral region reflects quantitative and qualitative changes in amides I, II in amino acids and proteins (1,700–1,500 cm-1), phosphodiester groups from nucleic acids and phospholipids (1,500–1,300 cm-1), and carbohydrate compounds (1,100–950 cm-1). PCA revealed separate clusters corresponding to production traceability relationships. Therefore, PCA can be used to distinguish between production in 2019, 2020, and 2021 based on different metabolite contents. PLS-DA showed a similar production traceability classification for the perilla seeds. In addition, this metabolic identification system can be used to rapidly select and classify useful perilla seed varieties.
들깨(Perilla frutescens)는 꿀풀과 일년생 초본 식물이며 종실이나 잎을 착유하여 기름을 사용하는 유료 작물로 소비되고 있다. 들깨는 생육기간이 짧고 지역적응성이 높아 타작물과의 혼작이나 윤작이 가능하다(Choung 2005, Lee et al. 2019, Lee et al. 2021).
들깨에 함유되어 있는 약 44%의 지질은 다양한 기능성이 알려져 있다. omega-3는 심혈관, 고혈압, 염증, 우울증 등의 예방 효과로 알려져 있으며 그 중 α-linolenic acid는 다양한 질병 예방에 효과가 있다(Gu et al. 2009, Lee et al. 2016). 또한, 불포화지방산인 linoleic acid와 oleic acid을 함유하고 있고, rosmarinic acid, luteolin 등의 phenolic compounds가 함유되어 있는 것으로 알려져 있다(Ghimire et al. 2017)
들깨 생산연도 판별은 생산자와 소비자 간에 유통질서 확립을 위해 필요한 시스템이다. 생산 연도에 따른 발아율과 특성 등이 연도 간 차이를 나타내고, 육안 식별이 어려움에 따라 생산연도 식별이 불가능하다. 또한, 동아시아를 중심으로 기름으로 소비되고 있는 유료 작물로 생산 연도에 따른 omega-3, linoleic acid와 oleic acid 등의 성분이 변화하기 때문에 생산연도 판별이 중요하다(Ghimire et al. 2017, Gu et al. 2009, Lee et al. 2016, Seong et al. 2015). 이처럼 생산시기에 따라 재배 조건과 환경변화를 통해 이차대사산물의 함량을 대사체 시료 간 양적, 질적 패턴 차이를 통해 생산 연도를 판별할 수 있다.
오믹스 기술의 발달은 분자생물학적 분석법들의 시간과 비용 절감이 이루어지고 있다. 대표적으로 Fourier transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (proton NMR) spectroscopy 및 Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) 등의 분석을 통해 시료 간의 대사체 물질 차이를 판별하고 대사체 마커 개발에 활용되고 있다(Krishnan et al. 2005). 특히, FT-IR spectroscopy을 이용한 대사체학 연구는 스펙트럼상에 amino acids (glutamic acids 1700-1750 cm-1, aspartic 1400-1430 cm-1, trytophan 1360 cm-1), proteins (α-Helix 1275 cm-1, Anti-parallel β-sheet 1235 cm-1), lipids/fatty acids (υ (C-C) 1100-800 cm-1), monosaccharides (α, β-glucose, β-fructose 898-847 cm-1) 등 시료간의 양적, 질적 차이를 다변량 통계분석을 이용한 품종 식별을 통해 구분할 수 있다(Gallardo-Velázquez et al. 2009, Schulz & Baranska 2007).
대사체학은 대사성분의 작용기작, 대사경로, 물질 탐색 등과 같은 기초학문적 활용을 통해 품종 식별, 원산지 판별, 유용성분 함량 예측, 재배연령 등의 진위 식별, 표준화와 안전성에 관련한 식품과 의약품 산업에 다양하게 활용되고 있다. 대사체 분석을 통한 데이터 활용은 유용 생물학적 정보 추출을 위해 principal component analysis (PCA), partial least square discriminant analysis (PLS-DA) 그리고 partial least squares (PLS) regression은 대표적인 단변량 통계분석 기법으로 사용된다. 이러한 FT-IR 분석 기술을 이용하여 미생물종의 유연관계를 식별, 돌연변이 선발, 고등식물의 종 구분, 두과작물 품종 식별 등에 활용이 가능하다고 보고된 바 있다(Chen et al. 1998, Goodacre et al. 1998, Kim et al. 2004, Song et al. 2012, Stewart et al. 1997, Timmins et al. 1998, Wenning et al. 2002).
따라서 본 연구는 FT-IR 스펙트럼 데이터의 다변량 통계분석 기법을 이용하여 들깨 생산 연도에 따른 신속한 구분 및 식별 체계를 확립하고자 한다.
시료 준비와 들깨 잎 재배조건
본 연구에서 생산연도 판별을 위해 사용된 들깨 종자는 국립식량과학원 남부작물부 밭작물개발과에서 2019~2021년도에 생산된 국산 들깨 38종을 분양 받아 사용하였다(Table 1). 들깨 종자는 수확 후 저온 창고에 4℃로 일정한 온도를 유지하며 보관하였다. 들깨 잎 재배를 위해 각각의 연도에 해당하는 4℃ 저온 창고에 보관중인 종자를 파종 전 날에 미리 실온에 꺼내 두었다. 가로 48 cm 세로 36 cm 높이 5.1 cm 48구 포트에 엽채류, 조미채소, 과채류, 기타채소 등에 주로 많이 사용하는 수라상토 1호(Heungnong, Korea)를 이용하여 상토재배법으로 온도 22℃, 습도 60%로 2주간 온실에서 재배 하였다. 들깨 잎은 뿌리를 제외하고 줄기와 잎을 포함한 지상부 전체를 수확하여 실험에 사용하였다.
실험에 사용된 재료는 파종 2주 후 들깨 잎을 채취하여 45℃ 건조기에 3일동안 건조 후 Wonder crusher WC-3를 이용하여 분말형태로 분쇄하였다. 분쇄된 시료는 -70°C 초저온냉동고에 보관하여 분석에 사용하였고, 품종별 3반복으로 진행하였다.
들깨 whole-cell 추출물 제조
들깨 잎 시료 분말 20 mg과 20% (v/v) methanol 용매 200 µL을 1.5 mL tube에 넣고 시료와 용액이 잘 섞이도록 vortexing 한 후 50℃ water bath에서 30분간 추출하였다. 시료를 잘 섞어주기 위해 5분 간격으로 vortexing 하였다. 추출된 시료는 13,000 rpm에 15분간 원심분리 후 새로운 1.5 mL tube에 상층액을 옮겼다. 추출물 찌꺼기를 완전히 제거하기 위해 다시 한번 13,000 rpm에 5분간 원심분리하여 상층액을 새로운 1.5 mL tube로 옮겼다. FT-IR 스펙트럼 분석에 사용하기 위해 추출된 추출물은 -20°C에서 냉동 보관하여 사용하였다(Song et al. 2014).
FT-IR 스펙트럼과 데이터 전처리 및 다변량 통계분석
FT-IR (Fourier transform infrared) 스펙트럼 조사는 Tensor II (Bruker Optics GmbH, Ettlingen, Germany)과 DTGS (deuterated triglycine sulfate) 검출기를 사용하였다(Song et al. 2014). 각각의 추출된 시료는 5 µL씩 5반복으로 384-well ZnSe plate에 분주하여 37℃ hot plate에서 약 20-30분간 건조한 후, HTS-XT (Bruker Optics GmbH) 고효율 자동화 장치를 이용하여 스펙트럼을 측정하였다. 스펙트럼은 4000-400 cm-1 범위에서 4 cm-1 간격으로 128회 반복하여 측정되었으며, 측정된 스펙트럼의 평균 스펙트럼을 분석에 사용하였다. Bruker 에서 제공하는 OPUS Lab (ver. 7.0, Bruker Optics Inc., U.S.A)를 사용하여 FT-IR 스펙트럼 조사 및 데이터 변환을 하였다. Baseline 교정을 위해 FT-IR 스펙트럼 분석 영역의 양 끝점(800-1800 cm-1)의 흡광도를 0으로 조정하였다. 실험상의 오차를 최소화하기 위하여 각 스펙트럼을 동일 면적으로 normalization을 실시하였으며, mean centering 과정을 거쳐 2차 미분하여 스펙트럼의 전처리 과정을 실시하였다(Song et al. 2014). 가공된 FT-IR 스펙트럼 데이터는 NIPALS 알고리즘(Wold 1966)을 이용하여 R 프로그램(version 3.1.2)을 사용하여 PCA (principal component analysis)와 PLS-DA (partial least squares discriminant)분석을 수행하였다(Fiehn et al. 2000, Song et al. 2020, Trygg et al. 2007).
들깨 잎의 FT-IR 스펙트럼 비교 분석
들깨 잎의 생산연도 식별을 위해 FT-IR 스펙트럼 데이터로부터 다변량 통계분석을 통해 들깨 잎의 생산 연도별 유연관계를 분석하였다. 각각의 품종들은 FT-IR 스펙트럼 1750-1500, 1500-1300, 1150-950 cm-1 상에서 특정적인 스펙트럼 패턴 변화가 나타났다(Fig. 1). FT-IR 스펙트럼의 특정적인 주요 부위는 각각 특정 화합물들을 나타내는데, 1750-1500 cm-1 부위에서는 주로 Amide I 과 II을 포함하는 P=O, C=O, N-H, C-C 그리고 C=C (아미노산 및 단백질계열의 화합물)들의 질적, 양적 정보를 나타내고, 1500-1300 cm-1 부위에서는 phosphodiester group을 포함한 CH2와 P=O (핵산 및 인지질)의 정보가 반영이 되고, 그리고 1100-950 cm-1 부위에서는 carbohydrates 계열의 C-O, C-C 그리고 C-O-C (단당류나 복합 다당류)를 포함하는 화합물들이 질적, 양적 정보가 반영되어 나타낸다(D’Souza et al. 2008, Dumas & Miller 2003, Lopez-Sanchez et al. 2010, Parker 1983, Wolkers et al. 2004, Yee et al. 2004). 이와 같이 FT-IR 스펙트럼 상의 들깨 잎이 함유하고 있는 지방산, 아미노산, 단백질 그리고 탄수화물계통의 화합물들의 질적, 양적 차이가 뚜렷하게 나타났다. 따라서, FT-IR 스펙트럼 분석을 이용한 주요 대사체의 질적 변화와 양적 변화를 분석하여 들깨 잎의 생산연도 판별 수단으로써 활용이 가능할 것이다.
FT-IR 스펙트럼 다변량 통계분석 및 대사체 수준 유연 관계 비교
들깨 잎의 FT-IR 스펙트럼 데이터를 이용하여 PCA 분석한 결과, PC 1과 2는 96.2%와 2.4%의 설명력을 각각 나타났다(Fig. 2A). 들깨 잎의 생산연도 차이는 우측에 2019년 생산 들깨가 위치하고, 2020년과 2021년도 생산 들깨가 좌측 하단과 상단에 위치하였다(Fig. 2A). 이처럼 들깨 잎의 생산 연도에 따라 서로 다른 분포를 나타내면서 생산 연도별 대사체 변화를 확인할 수 있었다. 아직까지 FT-IR 스펙트럼 데이터를 이용한 들깨 잎의 생산연도 판별 분석이 없었지만, 두과작물의 품종간 식별(Song et al. 2012), 배추 계통 식별을 통한 F1 판별(Ahn et al. 2016), 아티초크의 품종간 식별(Kim et al. 2016), 들깨 품종의 원산지 판별이 가능한 것으로 보고되었다(Yang et al. 2022). 따라서, 들깨 잎의 FT-IR 스펙트럼 데이터를 이용한 대사체 분석을 통해 생산연도 간의 차이와 유의성을 확인할 수 있다. 이상의 방법을 이용하여 들깨 잎의 품종 육성에 정확하고 빠른 선택을 할 수 있을 것이다.
들깨 잎의 FT-IR 스펙트럼 데이터 식별에 중요하게 작용한 FT-IR 스펙트럼 부위를 확인하였다(Fig. 2B). FT-IR loading value를 확인한 결과, 들깨 잎의 생산연도를 구분하기 위하여 PC1과 2를 결정하는 주요 FT-IR 부위는 1700-1500, 1500-1300 그리고 1100-950 cm-1 였다(Fig. 2B). FT-IR 스펙트럼상에서 차이를 나타냈던 부위(Fig. 1)와 loading value 상에서 나타난 부위가 일치하였고, 이 부위가 나타내는 아미노산 및 단백질계열의 화합물, 핵산 및 인지질 그리고 carbohydrates 계열의 질적, 양적 차이가 나타남을 의미하며 들깨 잎의 생산연도 판별에 중요한 역할을 하는 것으로 알 수 있었다(Schulz & Baranska 2007) (Fig. 2B).
PCA는 데이터의 variance를 최대한 유지하면서 dimension reduction 하려고 하는 반면에 PLS-DA는 사전에 그룹에 관한 정보를 미리 줌으로써 convariance를 최대한 유지하면서 분석하는 방법으로 PLS-DA 분석의 경우, PCA 분석 보다 들깨 잎의 생산연도 식별이 뚜렷하게 나타났다(Fig. 3). PLS-DA score plot을 보면 좌측에 2019년 생산 들깨가 우측 상단에 2020년 생산 들깨가 위치하고, 우측 하단에 2021년 생산 들깨가 위치하는 것을 확인할 수 있었다. 이상의 연구 결과는 FT-IR 스펙트럼 데이터를 이용한 생산연도 식별이 가능함을 알 수 있었다. 현재까지 FT-IR 스펙트럼 데이터를 이용하여 들깨 잎의 생산연도 식별이 보고되지 않았지만, 최근 다양한 대사체 분석을 이용한 품종 식별 사례가 보고되어 있다. 아열대 작물인 커피에서 대사체 분석을 통해 품종간에 차이를 분석하였고(Kim et al. 2018), 두과작물과 아티초크에서 FT-IR 스펙트럼을 이용한 품종간 대사체 분석에서 스펙트럼 상에서 질적, 양적 차이를 구분하여 품종 식별이 가능하다고 보고되었다(Kim et al. 2016, Song et al. 2012). 또한, 들깨 잎의 FT-IR 스펙트럼 데이터를 이용한 들깨 잎의 원산지 식별이 가능함이 보고되었다(Yang et al. 2022). 이처럼 FT-IR 스펙트럼의 특정 부위들은 들깨 잎에서도 중요하게 작용하여 생산연도 식별에 관여하는 것을 알 수 있었다. 또한, FT-IR 스펙트럼 데이터를 이용한 생산연도 식별은 대사물질의 유연관계가 차이를 보였고, 이러한 차이가 생산연도를 결정하는데 중요한 역할을 하는 것으로 판단된다.
따라서 본 연구에서 확립된 FT-IR 스펙트럼 데이터를 이용한 들깨 잎의 생산연도 판별 기술은 들깨 잎에 함유되어 있는 지표물질 탐색 없이 전체 대사물질에 상관성을 확인함으로써 생산연도에 따른 관계를 알 수 있어 재료 선발시 활용될 것으로 예상된다.
본 연구는 FT-IR 스펙트럼 데이터를 이용하여 들깨 잎의 대사체 수준에서 생산연도 판별을 실시하였다. FT-IR 스펙트럼 데이터는 다변량 통계분석을 통해 PCA와 PLS-DA 분석하였다. FT-IR 스펙트럼의 1,700-1,500, 1,500-1,300 그리고 1,100-950 cm-1 부위는 주로 아미노산, 핵산, 당, 지질 그리고 단백질을 나타낸다. 이러한 대사체를 이용해 PCA를 분석한 결과, 들깨 잎은 2019, 2020 그리고 2021년 생산연도에 따른 각각의 그룹을 형성하면서 생산연도 식별이 가능했다. 또한 생산연도에 따라 그룹을 형성하는 것을 PLS-DA에서도 볼 수 있었다. 따라서, FT-IR 스펙트럼 데이터의 다변량 통계분석을 통해 들깨 잎의 수준에서 생산연도간의 차이를 확인할 수 있었으며, 정확하고 빠른 식별을 통해 생산연도 식별에 이용이 가능할 것으로 판단된다.
본 논문은 농촌진흥청 연구사업(과제번호:PJ0160882023)의 지원에 의해 수행하였습니다.
Fig. 1
Representative FT-IR spectral from Perilla leaf of main cultivar. FT-IR spectral ranges showed quantitative information of protein/ amide I, II (1500-1700 cm-1), phosphodiester group (1300-1500 cm-1), and sugar compound (950-1100 cm-1).
kjbs-56-1-11-f1.jpg
Fig. 2
PCA score plot (A) and loading value plot (B) of PCA analysis from FT-IR data of Perilla leaf. Dotted shapes represent significant FT-IR spectral region for metabolic discrimination from year of production.
kjbs-56-1-11-f2.jpg
Fig. 3
PLS-DA score plot of FT-IR data from Perilla leaf. Dotted shapes represent significant FT-IR spectral region for metabolic discrimination from year of production.
kjbs-56-1-11-f3.jpg
Table 1
List of 38 Perilla frutescens used in this study.
Table 1
No. Name No. Name No. Name No. Name
1 Areum 11 Dasil 21 Ilyeop 31 Sangbaek
2 Baekjin 12 Dayu 22 Ipdeulkkae1 32 Saeyeopsil
3 Baekkang 13 Deulchan 23 Joim 33 Sangyeup
4 Baeksang 14 Deulhyang 24 Manbaek 34 Sodam
5 Bora 15 Deulsaem 25 Namcheon 35 Soim
6 Daesin 16 Donggeul1 26 Neulbora 36 Somirang
7 Daeyu 17 Donggeul2 27 Nulsaemi 37 Yeopsil
8 Daeyeop 18 Gwangim 28 Okdong 38 Yujin
9 Dami 19 Hwahong 29 Saebora
10 Danjo 20 Hyangim 30 Saebom
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Determination of Production Year Using Multivariate Statistical Analysis from FTIR Spectrum Data of Perilla Leaves
Korean. J. Breed. Sci.. 2024;56(1):11-18.   Published online March 1, 2024
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Determination of Production Year Using Multivariate Statistical Analysis from FTIR Spectrum Data of Perilla Leaves
Image Image Image
Fig. 1 Representative FT-IR spectral from Perilla leaf of main cultivar. FT-IR spectral ranges showed quantitative information of protein/ amide I, II (1500-1700 cm-1), phosphodiester group (1300-1500 cm-1), and sugar compound (950-1100 cm-1).
Fig. 2 PCA score plot (A) and loading value plot (B) of PCA analysis from FT-IR data of Perilla leaf. Dotted shapes represent significant FT-IR spectral region for metabolic discrimination from year of production.
Fig. 3 PLS-DA score plot of FT-IR data from Perilla leaf. Dotted shapes represent significant FT-IR spectral region for metabolic discrimination from year of production.
Determination of Production Year Using Multivariate Statistical Analysis from FTIR Spectrum Data of Perilla Leaves

List of 38 Perilla frutescens used in this study.

No. Name No. Name No. Name No. Name
1 Areum 11 Dasil 21 Ilyeop 31 Sangbaek
2 Baekjin 12 Dayu 22 Ipdeulkkae1 32 Saeyeopsil
3 Baekkang 13 Deulchan 23 Joim 33 Sangyeup
4 Baeksang 14 Deulhyang 24 Manbaek 34 Sodam
5 Bora 15 Deulsaem 25 Namcheon 35 Soim
6 Daesin 16 Donggeul1 26 Neulbora 36 Somirang
7 Daeyu 17 Donggeul2 27 Nulsaemi 37 Yeopsil
8 Daeyeop 18 Gwangim 28 Okdong 38 Yujin
9 Dami 19 Hwahong 29 Saebora
10 Danjo 20 Hyangim 30 Saebom
Table 1 List of 38 Perilla frutescens used in this study.