molecules Article Metabolomics Provides Quality Characterization of Commercial Gochujang (Fermented Pepper Paste) Gyu Min Lee, Dong Ho Suh, Eun Sung Jung and Choong Hwan Lee * Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea; [email protected] (G.M.L.); [email protected] (D.H.S.); [email protected] (E.S.J.) * Correspondence: [email protected]; Tel.: +82-2-2049-6177; Fax: +82-2-455-4291 Academic Editor: Marcello Iriti Received: 3 June 2016; Accepted: 8 July 2016; Published: 15 July 2016 Abstract: To identify the major factors contributing to the quality of commercial gochujang (fermented red pepper paste), metabolites were profiled by mass spectrometry. In principal component analysis, cereal type (wheat, brown rice, and white rice) and species of hot pepper (Capsicum annuum, C. annuum cv. Chung-yang, and C. frutescens) affected clustering patterns. Relative amino acid and citric acid levels were significantly higher in wheat gochujang than in rice gochujang. Sucrose, linoleic acid, oleic acid, and lysophospholipid levels were high in brown-rice gochujang, whereas glucose, maltose, and γ-aminobutyric acid levels were high in white-rice gochujang. The relative capsaicinoid and luteolin derivative contents in gochujang were affected by the hot pepper species used. Gochujang containing C. annuum cv. Chung-yang and C. frutescens showed high capsaicinoid levels. The luteolin derivative level was high in gochujang containing C. frutescens. These metabolite variations in commercial gochujang may be related to different physicochemical phenotypes and antioxidant activity. Keywords: gochujang; quality characterization; metabolomics; GC-TOF-MS; UHPLC-LTQ-ESI-IT- MS/MS; antioxidant activity 1. Introduction Food is essential to sustain life and has undergone continuous development worldwide for a long time. As the industrialization of food products has progressed over the past few decades, the public interest in health and well-being has also increased [1]. To meet these demands, application of scientific knowledge to the food industry has become necessary to control quality and safety as well as to maintain the beneficial components in commercial food products [2]. However, various factors contribute to food quality, such as biological origin, ecological conditions, microbiome, and processing methods. Particularly, fermented foods contain more complex factors and mechanisms than other food types. Gochujang (fermented red pepper paste) is a traditional food that has unique properties, including spicy flavor, pungency, sweet taste, and distinctive texture. It is rich in nutrient components, such as essential amino acids, fatty acids, organic acids, and sugars, which are generated from raw materials during fermentation [3]. Furthermore, gochujang has various functional and biological activities, such as anti-obesity [4,5], anti-tumor [6], and anti-cancer [7] effects. The general manufacturing process for commercial gochujang is complex because of the combination of various raw materials and the diverse conditions required for production, which are summarized in Figure1. Because of these complexities, it is difficult to control the quality of commercial gochujang. Thus, it is important to identify the major factors, as well as their mechanisms of action, affecting the quality of commercial gochujang. Molecules 2016, 21, 921; doi:10.3390/molecules21070921 www.mdpi.com/journal/molecules Molecules 2016, 21, 921 2 of 14 Molecules 2016, 21, 921 2 of 14 Figure 1. The schematic diagram of the general manufacturing process and the major raw ingredients Figure 1. The schematic diagram of the general manufacturing process and the major raw ingredients for commercial gochujang. * Additives; salt, starch syrup, garlic, onion, and soy powder are typically for commercialon the ingredient gochujang. label *of Additives; the products. salt, starch syrup, garlic, onion, and soy powder are typically on the ingredient label of the products. Metabolomics have been developed in numerous fields, including pharmacology, toxicology, plant physiology, food science, and nutrition [8–11]. Metabolomics techniques are very important Metabolomics have been developed in numerous fields, including pharmacology, toxicology, tools in food science in particular to ensure food safety, quality, and traceability [12]. For example, plant physiology,these methods food can science, be applied and to nutrition detect contaminants, [8–11]. Metabolomics pathogens, and techniques toxins in food are [13]; very to importantevaluate tools in foodthe science quality in of particular fruits or beverages to ensure based food on metabolite safety, quality, profiles and [14]; traceabilityand to monitor [12 metabolic]. For example, changes these methodsduring can bestorage applied and fermentation to detect contaminants,[15,16]. Metabolomi pathogens,cs approaches and are toxins actively in used food to [investigate13]; to evaluate the qualityfermented of fruits foods orsuch beverages as cheeses, basedwines, fermented on metabolite soy products, profiles and [probiotics14]; and [17]. to Metabolomics monitor metabolic approaches have been used to evaluate gochujang to confirm the bioactivities of extracts or specific changes during storage and fermentation [15,16]. Metabolomics approaches are actively used to compounds and to investigate the bioconversion of compounds during fermentation [3–7]. However, investigatefew studies fermented have examined foods such differences as cheeses, in metabolites wines, to fermented characterize soy commercial products, gochujang. and probiotics [17]. MetabolomicsIn this approaches study, the haveuntargeted been metabolite used to evaluate profiling gochujangof various commercial to confirm gochujang the bioactivities preparations of extracts or specificwas compounds performed using and GC-TOF-MS to investigate and the ultrahigh bioconversion performance of compoundsliquid chromatography-linear during fermentation trap [3–7]. However,quadrupole-ESI-ion few studies have trap-MS/MS examined (UHPLC-LTQ-ESI-IT- differences in metabolitesMS/MS) with to characterize multivariate statistical commercial analysis gochujang. In thisto investigate study, the the untargetedmajor factors metabolitecontributing profilingto the metabolite of various state and commercial physicochemical gochujang quality preparationsof the final commercial form of gochujang. In addition, we propose specific metabolites as quality markers for was performed using GC-TOF-MS and ultrahigh performance liquid chromatography-linear trap commercial gochujang. quadrupole-ESI-ion trap-MS/MS (UHPLC-LTQ-ESI-IT-MS/MS) with multivariate statistical analysis to investigate2. Results the major factors contributing to the metabolite state and physicochemical quality of the final commercial form of gochujang. In addition, we propose specific metabolites as quality markers 2.1. Metabolite Profiling of Commercial Gochujang Samples for commercial gochujang. To investigate the factors affecting the differences among the final products of a diverse 2. Resultsgroup of gochujang samples (Figure 1 and Table 1) and to characterize the metabolites affecting the physicochemical quality of commercial gochujang, multivariate statistical analysis was performed using 2.1. Metabolitethe GC-TOF-MS Profiling and of CommercialUHPLC-LTQ-ESI-IT Gochujang-MS/MS Samples data sets (Figure 2). The variance of each PCs of the three-dimensional (3D) PCA score plots were PC1 (12.3%), PC2 (11.9%), and PC3 (7.6%) (Figure 2A) and ToPC1 investigate (9.9%), PC2 the (6.7%), factors and affectingPC3 (6.3%) the(Figure differences 2B). Although among the total the variances final products are low, the of aexplained diverse group of gochujangvariances samples (R2) values (Figure of PCA models1 and were Table over1) 0.4 and (R2X to(cum)characterize = 0.582 and 0.477), the reasonably. metabolites And the affecting distinct the physicochemical quality of commercial gochujang, multivariate statistical analysis was performed using the GC-TOF-MS and UHPLC-LTQ-ESI-IT-MS/MS data sets (Figure2). The variance of each PCs of the three-dimensional (3D) PCA score plots were PC1 (12.3%), PC2 (11.9%), and PC3 (7.6%) (Figure2A) and PC1 (9.9%), PC2 (6.7%), and PC3 (6.3%) (Figure2B). Although the total variances are 2 2 low, the explained variances (R ) values of PCA models were over 0.4 (R X(cum) = 0.582 and 0.477), reasonably. And the distinct patterns for commercial gochujang are shown based on their raw materials Molecules 2016, 21, 921 3 of 14 Moleculespatterns2016, 21, 921for commercial gochujang are shown based on their raw materials in the 3D PCA 3score of 14 plots. As shown in Figure 2, the samples were mainly divided into two groups by cereal type, gochujang Molecules 2016, 21, 921 3 of 14 made with wheat (WG) and gochujang made with rice (RG). Within the cluster of RG, there was in the 3D PCA score plots. As shown in Figure2, the samples were mainly divided into two groups patternsdifferentiation for commercial into groups gochujang of goarechujang shown based prepared on their using raw materials brown rice in the (RbG) 3D PCA and score white plots. rice (RwG). In by cereal type, gochujang made with wheat (WG) and gochujang made with rice (RG). Within the Asthe shown expanded in Figure figures, 2, the samplesthe two were clusters mainly of dividedRbG and into RwG two groups were separatedby cereal type, according gochujang to hot pepper cluster of RG, there was differentiation into groups of gochujang prepared using