[논문]Volatile Metabolic Markers for Monitoring Pectobacterium carotovorum subsp. carotovorum Using Headspace Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry

Yang, Ji-Su; Lee, Hae-Won; Song, Hyeyeon; Ha, Ji-Hyoung

doi:10.4014/jmb.2009.09028

Volatile Metabolic Markers for Monitoring Pectobacterium carotovorum subsp. carotovorum Using Headspace Solid-Phase Microextraction Coupled with Gas Chromatography-Mass Spectrometry 원문보기

Journal of microbiology and biotechnology, v.31 no.1, 2021년, pp.70 - 78

Yang, Ji-Su (Hygienic Safety and Analysis Center, World Institute of Kimchi) , Lee, Hae-Won (Hygienic Safety and Analysis Center, World Institute of Kimchi) , Song, Hyeyeon (Hygienic Safety and Analysis Center, World Institute of Kimchi) , Ha, Ji-Hyoung (Hygienic Safety and Analysis Center, World Institute of Kimchi)

Abstract ▼ AI-Helper

Identifying the extracellular metabolites of microorganisms in fresh vegetables is industrially useful for assessing the quality of processed foods. Pectobacterium carotovorum subsp. carotovorum (PCC) is a plant pathogenic bacterium that causes soft rot disease in cabbages. This microbial species in plant tissues can emit specific volatile molecules with odors that are characteristic of the host cell tissues and PCC species. In this study, we used headspace solid-phase microextraction followed by gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS) to identify volatile compounds (VCs) in PCC-inoculated cabbage at different storage temperatures. HS-SPME-GC-MS allowed for recognition of extracellular metabolites in PCC-infected cabbages by identifying specific volatile metabolic markers. We identified 4-ethyl-5-methylthiazole and 3-butenyl isothiocyanate as markers of fresh cabbages, whereas 2,3-butanediol and ethyl acetate were identified as markers of soft rot in PCC-infected cabbages. These analytical results demonstrate a suitable approach for establishing non-destructive plant pathogen-diagnosis techniques as alternatives to standard methods, within the framework of developing rapid and efficient analytical techniques for monitoring plant-borne bacterial pathogens. Moreover, our techniques could have promising applications in managing the freshness and quality control of cabbages.

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문제 정의

Therefore, in this study, we used automated HS-SPME-GC–MS-based extracellular metabolomics as an analytical approach for identifying VCs related to PCC in cabbage. The study was conducted to determine whether HS-SPME-GC-MS could differentiate VCs emitted from cabbage samples under different conditions by measuring their headspace volatiles. We also characterized the VC profiles and determined the specific volatile metabolic markers of different sample groups (fresh cabbage without external infection symptoms and artificially infected cabbage showing external infection symptoms, at different storage temperatures) using GC-MS at the laboratory scale.

제안 방법

Additionally, it has been shown that the important features identified by PLS-DA through a typical multivariate analysis, such as VIP values, are effective for determining potential biomarkers [32]. Based on the PLS-DA model, we further analyzed the VIP values to determine the metabolites with VIP scores greater than 1, which represented important differential VCs. The results of volatile biomarker analysis indicated that 2, 3-butanediol, 3-butenyl isothiocyanate, ethyl acetate, and 4-ethyl-5-methylthiazole were important metabolites for defining the PLS-DA model that distinguished the PCC-infected and non-infected cabbage samples (Fig.
The findings agree with those of previous studies on the effects of PCC on pectate lyase activity, which is involved in the maceration and soft rotting of plant tissue [30]. The study reported the thermodependency of pectate lyase activity, in addition to a significant association between the influence of temperature on pectate lyase activity and soft rot. Remarkably, the groups of PCC-inoculated samples stored at 20℃ and 30℃ and the remaining groups (fresh cabbage samples stored at 0℃, 10℃, 20℃, and 30℃) revealed completely contrasting behaviors.
pekinensis) contaminated with PCC are limited. Therefore, in this study, we used automated HS-SPME-GC–MS-based extracellular metabolomics as an analytical approach for identifying VCs related to PCC in cabbage. The study was conducted to determine whether HS-SPME-GC-MS could differentiate VCs emitted from cabbage samples under different conditions by measuring their headspace volatiles.
This study focused on detecting soft rot in cabbages by identifying volatile metabolic marker compounds for the disease using HS-SPME combined with GC-MS analysis. In recent years, the random forest (RF), a machine learning method, has been extensively used in the field of metabolomics for biomarker discovery [31, 32].
6A). Using RF analysis, the features were ranked by a measure of permutation importance of the predictor’s classification of individual samples based on the mean decrease in accuracy. As shown in Fig.

대상 데이터

25 μm film thickness; Agilent). Helium (99.999% or more) was used as the carrier gas, and the flow rate was set to 1 ml/min. The temperature of the inlet set to splitless mode was 250℃.
The degree of difference in VCs between the experimental groups was identified by variable importance in projection (VIP) scores estimated as a weighted sum of squares of the PLS loadings, considered the amount of explained y variation for each dimension. Three experimental replications were performed for each cabbage sample.

데이터처리

The main VCs identified in the stored cabbage samples are listed in Table S1. The difference in the VCs of the two groups of cabbage samples at each storage temperature was analyzed using a clustering heatmap and comparable VCs, selected by a t-test (p<0.01) (Fig. 4). As shown in Fig.

이론/모형

Approximately 20–25 ml of tryptic soy agar at 55℃ was poured on the plates. The Petri dishes were incubated at 28℃ for 20 h, and the PCC colonies appearing on the plates were counted using the standard plate count method and expressed as CFU/g. Additionally, we quantified the viable PCC cells in cabbage samples by quantitative reverse transcriptase PCR assays combined with an intercalating propidium monoazide dye [23].

성능/효과

Interestingly, our results indirectly demonstrate that a suitable storage temperature required to maintain cabbage quality is lower than 10℃. For the 3D score plot, the first three PCs accounted for 97.5% of the total data variability among the Bpellets, fresh cabbages, and PCC-inoculated cabbages (Fig. 2B). The obtained PCs clearly distinguished the experimental data on a hyperplane that differentiated the fresh cabbages and PCC-inoculated cabbages.
Based on the PLS-DA model, we further analyzed the VIP values to determine the metabolites with VIP scores greater than 1, which represented important differential VCs. The results of volatile biomarker analysis indicated that 2, 3-butanediol, 3-butenyl isothiocyanate, ethyl acetate, and 4-ethyl-5-methylthiazole were important metabolites for defining the PLS-DA model that distinguished the PCC-infected and non-infected cabbage samples (Fig. 6A). Using RF analysis, the features were ranked by a measure of permutation importance of the predictor’s classification of individual samples based on the mean decrease in accuracy.

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