Proteomic evaluation of kimchi, a traditional Korean fermented vegetable, and comparison of kimchi manufactured in China and Korea.

Kimchi is a traditional Korean fermented vegetable, which is also widely consumed in Japan and China. However, little is known about the kimchi proteome. In this study, Korean and Chinese kimchi proteomes were evaluated by shotgun proteomics. A total of 250 proteins were annotated, and 29 of these were expressed at > 1% of the average relative abundance. Discrimination of the geographical origins of Korean and Chinese kimchi samples was possible using multivariate analysis of the proteomic data, and 23 proteins were expressed differently between the two types (p < 0.001), and represent possible markers to discriminate between Chinese and Korean kimchi. This study provides important insights into the kimchi proteome and illustrates the proteomic differences caused by geographical origin.

Determination of macro, micro, trace essential, and toxic elements in Garcinia cambogia fruit and its anti-obesity commercial products.

BACKGROUND: Garcinia (Clusiaceae) species are traditionally used as flavoring agents in curries and to cure several human health complications. This study investigated 31 macro, micro, and trace elements in microwave-assisted digested samples of Garcinia cambogia fruit and its anti-obesity commercial products by inductively coupled plasma-optical emission spectroscopy (ICP-OES) and inductively coupled plasma-mass spectrometric (ICP-MS) techniques. The methods were also validated using the coefficient of determination (R2 ), limits of detection and quantification (LOD, LOQ), precision (CV%), analysis of certified reference materials, spiking recovery experiments, and participation in an accredited laboratory proficiency test organized by Food Analysis Performance Assessment Scheme (FAPAS). RESULTS: Quality assurance confirmed that the methods were efficient and in accordance with criteria set by the Association of Official Analytical Chemists (AOAC). In the elemental analysis, the analyzed macro, micro, and trace essential elements were present in appreciable concentrations, which could meet the human nutritional requirements. Traces of toxic elements were within safe limits. CONCLUSION: From the results of the current study, the fruit and its commercial products could be considered potential sources of mineral elements without posing any threats to consumers. © 2018 Society of Chemical Industry.

Determination of macro, micro and trace elements in citrus fruits by inductively coupled plasma-optical emission spectrometry (ICP-OES), ICP-mass spectrometry and direct mercury analyzer.

BACKGROUND: Citrus fruits are widely consumed around the world. Several elements are known to have important physiological functions in living things, whereas others are reported to have toxic effects. This study was aimed to analyze the concentration of macro, micro and trace elements in citrus fruit varieties. Samples were acid digested using a microwave system and analyzed for macro elements by inductively coupled plasma-optical emission spectrometry (ICP-OES) and for micro and trace elements by inductively coupled plasma-mass spectrometry (ICP-MS). The application of analytical techniques was analyzed by determining method validation parameters including limits of detection and quantification, linearity, precision, spiking experiments and analyzing certified reference material (NIST CRM-1570a), Spinach leaves. RESULTS: The elemental levels of citrus fruits were found largely to be dependent upon the type of analyzed samples. Among nutritionally important elements, citrus fruits were good sources of potassium (95.13-270.4 mg kg-1 ), calcium (10.57-75.29 mg kg-1 ), zinc (466.5-1611 µg kg-1 ) and manganese (35.35-1902 µg kg-1 ). The concentrations of toxic elements (Pb, Cd, As, Al, Hg) were very low. CONCLUSION: In general, the concentrations of analyzed elements were within the critical levels specified by the Food and Agriculture Organization/World Health Organization and Food and Nutrition Board USA. Linear discriminant analysis successfully separated the samples into clear groups with 93.9% correct classification. © 2018 Society of Chemical Industry.

Dietary intake assessment of macro, trace, and toxic elements via consumption of kimchi in South Korea.

BACKGROUND: Kimchi, a Korean food, is now produced and consumed in many other countries. In this study, the macro, trace, and toxic element content in varieties of kimchi from Korea and China was evaluated. A health-risk assessment, involving the calculation of estimated dietary intake values, was undertaken. To discriminate among samples with different origins, linear discriminant analysis (LDA) and principal component analysis (PCA) were applied. RESULTS: The analytical methods used for analyses were successfully validated. In the quantification of elements, no significant differences were detected in the concentrations of the elements that were analyzed. Based on the fresh weight of kimchi, among the macro elements, the concentrations of Na and K were relatively high, with values of 4181.9-9919.8 mg kg-1 and 1661.8-6623.5 mg kg-1 , respectively. Among the trace elements that were analyzed, zinc in Korean samples, and strontium in Chinese samples showed the highest concentrations, at 2.67 and 2.98 mg kg-1 , respectively. Concentration levels of the toxic trace elements were within permissible limits. The amount of daily intake and provisional daily intake of each element calculated based on kimchi intake confirmed its safety for consumption. Linear discriminant analysis and PCA successfully discriminated among the samples of Chinese and Korean origin. CONCLUSION: The provisional daily intake of the toxic elements was within provisional tolerable daily intake values. The normal intake of kimchi could not pose a threat upon consumption. © 2019 Society of Chemical Industry.

Temperature impact on microbial and metabolic profiles in kimchi fermentation.

Kimchi is a traditional Korean fermented food and harbors diverse bacteria. Therefore, proper temperature management contributes to the fermentation and preservation of kimchi. In this study, we explored fermentation temperature influences the bacterial composition and metabolite variations in kimchi, employing pyrosequencing for bacterial community analysis and mass spectrometry for metabolite profiling. Elevated temperatures within the range of 10-15 °C had a significant impact on the community of lactic acid bacteria (LAB) compared to 4 °C, leading to increased bacterial diversity and richness. We observed a significant increase in Lactiplantibacillus plantarum and Latilactobacillus sakei, alongside a reduction in Lactococcus lactis, during fermentation at 10-15 °C. These changes occurred within a similar pH range across different kimchi fermentation periods. We performed a liquid extraction via the acetonitrile method, which involved the collection of kimchi samples, and performed LC-MS analysis. Our analysis revealed approximately 5000 metabolites. Notably, we observed a significant increase in metabolite counts, with 3048 metabolites increasing at 10 °C and 2853 metabolites exhibiting a similar trend at 15 °C. Metabolite analysis showed an increase in lactic and succinic acid with increased glucose and sucrose consumption at 10 and 15 °C. These results indicated that elevated temperatures significantly influenced the glycolysis and tricarboxylic acid cycle, leading to increased acidity during the fermentation process. These findings show the crucial role played by temperature in controlling the fermentation process, thereby influencing the bacterial succession and the resulting flavor and taste of the product. Therefore, proper temperature management can effectively control kimchi fermentation and yield the desired sensory properties.

Exploring the influence of garlic on microbial diversity and metabolite dynamics during kimchi fermentation.

Garlic (Allium sativum) is a key ingredient in Korean cuisine, particularly in the preparation of kimchi, contributing to its flavor and taste. Garlic has been a potential resource for lactic acid bacteria (LAB) in kimchi. However, the mechanism by which it influences microbial diversity and metabolite production is unclear. This study investigated the effect of garlic on the bacterial composition of and metabolite changes in kimchi. To achieve this, four separate batches of kimchi were prepared with varying garlic concentrations (w/w): 0 %, 1 %, 2 %, and 4 %, and the bacterial communities and metabolite production were monitored. In the early stages of fermentation, the count of LAB, operational taxonomic units (OTUs), and Shannon index increased linearly with the increase in garlic content. This indicated that garlic is a rich resource and contributes to the diversity of LAB during kimchi fermentation. Compared with the kimchi samples with a lower garlic content, those with a high garlic content (≥2 %) exhibited increased abundance of Lactobacillus and Leuconostoc as well as noticeable differences in functional diversity, including carbohydrate, amino acid, and energy metabolisms. Correlation analysis between sugars, organic acids, and predominant LAB in the garlic-containing kimchi samples suggested that in kimchi samples with high garlic content, LAB played a significant role in the fermentation process by metabolizing sugars and producing organic acids. Overall, this study demonstrated that the addition of garlic has a positive impact on the bacterial diversity and metabolite production during kimchi fermentation, potentially affecting the fermentation process and flavor profile of kimchi.

Rapid analysis of insecticidal metabolites from the entomopathogenic fungus Beauveria bassiana 331R using UPLC-Q-Orbitrap MS.

Beauveria bassiana, a representative entomopathogenic fungus, is increasingly being utilized as an eco-friendly pest management alternative to chemical insecticides. This fungus produces a range of insecticidal secondary metabolites that act as antimicrobial and immunosuppressive agents. However, detailed qualitative and quantitative analysis related to these compounds remains scarce, we developed a method for the rapid analysis of these metabolites. Eight secondary metabolites (bassianin, bassianolide, beauvericin, beauveriolide I, enniatin A, A1, and B, and tenellin) were efficiently extracted when B. bassiana-infected Tenebrio molitor larvae were ground in 70% EtOH extraction solvent and subsequently subjected to ultrasonic treatment for 30 min. The eight metabolites were rapidly and simultaneously analyzed using ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS). Bassianolide (20.6-51.1 µg/g) and beauvericin (63.6-109.8 µg/g) were identified as the main metabolites in B. basssiana-infected larvae, indicating that they are likely major toxins of B. bassiana. Validation of the method exhibited recovery rates in the range of 80-115% and precision in the range of 0.1-8.0%, indicating no significant interference from compounds in the matrix. We developed a method to rapidly analyze eight insecticidal metabolites using UPLC-Q-Orbitrap MS. This can be extensively utilized for detecting and producing insecticidal fungal secondary metabolites.

Valorization of Cabbage Waste as a Feedstock for Microbial Polyhydroxyalkanoate Production: Optimizing Hydrolysis Conditions and Polyhydroxyalkanoate Production.

Establishing a platform for the bioconversion of waste resources into value-added compounds is critical for achieving a sustainable and eco-friendly economy. Herein, we produced polyhydroxyalkanoate via microbial fermentation using cabbage waste as a feedstock and metabolically engineered Escherichia coli. For this, the hydrolysis conditions of cabbage waste were optimized by focusing on parameters such as substrate and enzyme concentrations to enhance the saccharification efficiency. The phaABC operon, which encodes key enzymes responsible for polyhydroxyalkanoate biosynthesis in Ralstonia eutropha H16, was overexpressed in E. coli. Using cabbage hydrolysate as the feedstock, this engineered E. coli strain could produce poly(3-hydroxybutyrate) with a polymer content of 26.0 wt % of dry cell weight. Moreover, malic acid in cabbage hydrolysate significantly enhanced poly(3-hydroxybutyrate) production; the addition of 0.5 g/L malic acid markedly increased poly(3-hydroxybutyrate) content by 59.9%. This study demonstrates the potential of cabbage waste as a promising raw material for the microbial production of polyhydroxyalkanoate.

Utilization of coffee waste as a sustainable feedstock for high-yield lactic acid production through microbial fermentation.

Lactic acid is an important industrial precursor; however, high substrate costs are a major challenge in microbial fermentation-based lactic acid production. Coffee waste is a sustainable feedstock alternative for lactic acid production via microbial fermentation. Herein, the feasibility of coffee waste as a feedstock was explored by employing appropriate pretreatment methods and optimizing enzyme combinations. Coffee waste pretreatment with hydrogen peroxide and acetic acid along with a combination of Viscozyme L, Celluclast 1.5 L, and Pectinex Ultra SP-L achieved the 78.9 % sugar conversion rate at a substrate concentration of 4 % (w/v). Lactiplantibacillus plantarum WiKim0126-induced fermentation with a 4 % solid loading yielded a lactic acid concentration of 22.8 g/L (99.6 % of the theoretical maximum yield) and productivity of 0.95 g/L/h within 24 h. These findings highlight the viability of coffee waste as an eco-friendly resource for sustainable lactic acid production.

Transcriptional analysis of the molecular mechanism underlying the response of Lactiplantibacillus plantarum to lactic acid stress conditions.

Lactic acid bacteria (LAB) present various benefits to humans; they play key roles in the fermentation of food and as probiotics. Acidic conditions are common to both LAB in the intestinal tract as well as fermented foods. Lactiplantibacillus plantarum is a facultative homofermentative bacterium, and lactic acid is the end metabolite of glycolysis. To characterize how L. plantarum responds to lactic acid, we investigated its transcriptome following treatment with hydrochloride (HCl) or dl-lactic acid at an early stage of growth. Bacterial growth was more attenuated in the presence of lactic acid than in the presence of HCl at the same pH range. Bacterial transcriptome analysis showed that the expression of 67 genes was significantly altered (log2FC > 2 or < 2). A total of 31 genes were up- or downregulated under both conditions: 19 genes in the presence of HCl and 17 genes in the presence of dl-lactic acid. The fatty acid synthesis-related genes were upregulated in both acidic conditions, whereas the lactate racemization-related gene (lar) was only upregulated following treatment with dl-lactic acid. In particular, lar expression increased following l-lactic acid treatment but did not increase following HCl or d-lactic acid treatment. Expression of lar and production of d-lactic acid were investigated with malic and acetic acid; the results revealed a higher expression of lar and production of d-lactic acid in the presence of malic acid than that in the presence of acetic acid.

Rapid Quantitative Analysis of Metabolites in Kimchi Using LC-Q-Orbitrap MS.

Kimchi is a traditional Korean salted spontaneous lactic acid bacteria (LAB)-fermented food made using various vegetables. Organic acids, free sugars, and amino acids are key metabolites produced during LAB fermentation that determine the taste and quality of kimchi. However, each metabolite is typically analyzed using an independent analytical method, which is time-consuming and expensive. Therefore, in this study, we developed a method based on LC-Q-Orbitrap MS using which 20 types of representative fermented kimchi metabolites were selected and simultaneously analyzed within 10 min. The established method was validated, and its detection and quantification limits, linearity, precision, and accuracy were found to satisfy the Association of Official Agricultural Chemists (AOAC) validation guidelines. The 20 metabolites were simultaneously extracted from kimchi with different degrees of fermentation and quantitatively analyzed using LC-Q-Orbitrap MS. These results were analyzed using linear discriminant analysis and heat mapping, and the metabolites were grouped into early, middle, and late stages of fermentation. Malic acid (6.518-7.701 mMol) was only present in the initial stage of fermentation, and l-phenylalanine rapidly increased from the middle stage (2.180 mMol) to late stage (4.770 mMol). Lactic acid, which is representative of the sour taste of kimchi, was detected in the middle stage and increased rapidly up to 74.452 mMol in the late stage. In summary, in this study, 20 major kimchi metabolites were accurately analyzed within 10 min and grouped based on the degree of fermentation. Therefore, the method established in this study accurately and rapidly provides information on kimchi consumption and fermentation that could be highly valuable to the kimchi industry and kimchi consumers.

Quantification and health risk assessment of ochratoxin A in dried fruit, spices, and coffee.

Ochratoxin A (OTA) is a stable toxin produced by fungal strains of Aspergillus and Penicillium. It is commonly found in a variety of food products, including dried fruit, coffee, and spices, raising concerns about their safety. This study was aimed to quantify OTA levels in different food products using HPLC with fluorescence detection. The pre-treatment process was optimised by employing immunoaffinity columns with Tween 20 to effectively remove interfering substances. An analytical method was developed, validated, and applied for OTA analysis in dried fruit, spices, and coffee samples. The validation procedure included determining detection and quantification limits, linearity, precision, and accuracy, as per the criteria specified by AOAC International. The validated method was successfully applied for OTA analysis in the selected food samples. Furthermore, health risk assessment was conducted based on the average intake and body weight of the Korean population. From the results, concentrations of OTA in the samples were found to be very low and therefore concluded not to pose significant threats to consumer health.

Elemental Analysis of Kimchi Cabbage Leaves, Roots, and Soil and Its Potential Impact on Human Health.

In view of their rich mineral content and flavor, kimchi cabbage leaves and roots have high nutritional and medicinal values. In this study, we quantified major nutrient (Ca, Cu, Fe, K, Mg, Na, and Zn), trace (B, Be, Bi, Co, Ga, Li, Ni, Se, Sr, V, and Cr), and toxic (Pb, Cd, Tl, and In) elements in kimchi cabbage cultivation soil, leaves, and roots. The analysis method relied on inductively coupled plasma-optical emission spectrometry for major nutrient elements and inductively coupled plasma-mass spectrometry for trace and toxic elements and complied with the Association of Official Analytical Chemists (AOAC) guidelines. Kimchi cabbage leaves and roots featured high contents of K, B, and Be, while the contents of all toxic elements in all samples were below the WHO-stipulated threshold values and therefore did not pose any health risks. The distribution of elements was characterized by heat map analysis and linear discriminant analysis to reveal independent separation according to the content of each element. The analysis confirmed that there was a difference in content between the groups and that each group was independently distributed. This study may contribute to a better understanding of the complex relationships between plant physiology, cultivation condition, and human health.

Nondestructive classification of soft rot disease in napa cabbage using hyperspectral imaging analysis.

Identification of soft rot disease in napa cabbage, an essential ingredient of kimchi, is challenging at the industrial scale. Therefore, nondestructive imaging techniques are necessary. Here, we investigated the potential of hyperspectral imaging (HSI) processing in the near-infrared region (900-1700 nm) for classifying napa cabbage quality using nondestructive measurements. We determined the microbiological and physicochemical qualitative properties of napa cabbage for intercomparison of HSI information, extracted HSI characteristics from hyperspectral images to predict and classify freshness, and established a novel approach for classifying healthy and rotten napa cabbage. The second derivative Savitzky-Golay method for data preprocessing was implemented, followed by wavelength selection using variable importance in projection scores. For multivariate data of the classification models, partial least square discriminant analysis (PLS-DA), support vector machine (SVM), and random forests were used for predicting cabbage conditions. The SVM model accurately distinguished the cabbage exhibiting soft rot disease symptoms from the healthy cabbage. This study presents the potential of HSI systems for separating soft rot disease-infected napa cabbages from healthy napa cabbages using the SVM model, especially under the most effective wavelengths (970, 980, 1180, 1070, 1120, and 978 nm), prior to processing. These results are applicable to industrial multispectral images.

Efficient Citrus (Citrus unshiu) Byproduct Extract-Based Approach for Lactobacillus sakei WiKim31 Shelf-Life Extension.

Lactic acid bacteria produce various bioactive compounds widely used in human healthcare. However, studies on cryoprotective agents for the efficient storage of lactic acid bacteria after freeze-drying are still lacking. Here, we report the shelf-life extension effects of a highly efficient and eco-friendly cryoprotective agent and a cold adaptation method on Lactobacillus sakei WiKim31. Cold adaptation of L. sakei WiKim31 increased exopolysaccharide expression in response to abiotic stress. As a possible cryoprotective agent, the citrus byproduct (CP) contains a variety of sugars, amino acids, and cations, exhibiting high antioxidant activity. L. sakei WiKim31 powders formulated with CP or a mixture of soy powder (SP) and CP exhibited high cell viability at 58.3 and 76.3%, respectively, after 56 days of storage. These results indicate that CP can be efficiently used as a novel cryoprotective agent either alone or in combination with SP to improve the storage conditions of L. sakei WiKim31 and preserve it longer.

In Vitro and In Vivo Antibacterial Activity of Serratamid, a Novel Peptide-Polyketide Antibiotic Isolated from Serratia plymuthica C1, against Phytopathogenic Bacteria.

A new hybrid non-ribosomal peptide-polyketide antibiotic (serratamid) for phytoprotection was isolated from the ethyl acetate layer of tryptic soy agar culture of the soil bacterium Serratia plymuthica C1 through bioassay-guided fractionation. Its chemical structure was elucidated using instrumental analyses, such as mass and nuclear magnetic resonance spectrometry. Serratamid showed antibacterial activity against 15 phytopathogenic bacteria, with minimum inhibitory concentration (MIC) values ranging from 0.244 to 31.25 µg/mL. In vitro, it displayed strong antibacterial activity against Ralstonia solanacearum and four Xanthomonas spp., with MIC values (0.244-0.488 µg/mL) superior to those of streptomycin sulfate, oxolinic acid, and oxytetracycline. Further, serratamid and the ethyl acetate layer of S. plymuthica C1 effectively reduced bacterial wilt caused by R. solanacearum on tomato seedlings and fire blight caused by Erwinia on apple fruits in a dose-dependent manner. These results suggest that serratamid is a promising candidate as a potent bactericide for controlling bacterial diseases.

Determination of Toxic Elements and Arsenic Species in Salted Foods and Sea Salt by ICP-MS and HPLC-ICP-MS.

Toxic elements (Cd, Pb, and As) accumulate into the environment by industrialization and natural phenomena and then pass to organisms. Analysis of toxic elements in food must be accurately carried out on a regular basis so as to avoid any adverse impact. Salted foods are difficult samples and accurate analysis of As is not easy due to salt interference. In this study, analysis of As was carried without influence of salts in three types of salted foods via an analytical method, which was validated using spiking recovery experiments and by analyzing certified reference materials. As a result, toxic elements were detected in all samples but none of these exceeded the World Health Organization recommended limits. Among the As species, arsenobetaine (AsB) was the most abundant, while inorganic As was below the detection limit in all samples. All the analyzed salted food samples appeared to be safe for consumption. In addition, the analysis of sea shrimp, freshwater shrimp, and seawater verified As bioaccumulation in these organisms from the environment.

Nematicidal Activity of Cyclopiazonic Acid Derived From Penicillium commune Against Root-Knot Nematodes and Optimization of the Culture Fermentation Process.

Among 200 fungal strains isolated from the soil, only one culture filtrate of Aspergillus flavus JCK-4087 showed strong nematicidal activity against Meloidogyne incognita. The nematicidal metabolite isolated from the culture filtrate of JCK-4087 was identified as cyclopiazonic acid (CPA). Because JCK-4087 also produced aflatoxins, six strains of Penicillium commune, which have been reported to be CPA producers, were obtained from the bank and then tested for their CPA productivity. CPA was isolated from the culture filtrate of P. commune KACC 45973. CPA killed the second-stage juveniles of M. incognita, M. hapla, and M. arearia with EC50-3 days 4.50, 18.82, and 60.51 µg mL-1, respectively. CPA also significantly inhibited egg hatch of M. incognita and M. hapla after a total of 28 days of treatment with the concentrations > 25 µg mL-1. The enhancement of CPA production by P. commune KACC 45973 was explored using an optimized medium based on Plackett-Burman design (PBD) and central composite design (CCD). The highest CPA production (381.48 µg mL-1) was obtained from the optimized medium, exhibiting an increase of 7.88 times when compared with that from potato dextrose broth culture. Application of the wettable power-type formulation of the ethyl acetate extract of the culture filtrate of KACC 45973 reduced gall formation and nematode populations in tomato roots and soils under greenhouse conditions. These results suggest that CPA produced by P. commune KACC 45973 can be used as either a biochemical nematicide or a lead molecule for developing chemical nematicides to control root-knot nematodes.

Deciphering the Relationship Between Cycloheximides Structures and Their Different Biological Activities.

Streptomyces species are the most important sources of antibacterial, antifungal, and phytotoxic metabolites. In this study, cycloheximide (CH) and acetoxycycloheximide (ACH) were isolated from the fermentation broth of Streptomyces sp. JCK-6092. The antifungal and phytotoxic activities of the two compounds (CH and ACH) and a cycloheximide derivative, hydroxycycloheximide (HCH), were compared. CH exhibited the strongest antagonistic activity against all the true fungi tested, followed by ACH and HCH. However, both CH and ACH displayed similar mycelial growth inhibitory activities against several phytopathogenic oomycetes, and both were more active than that of HCH. Disparate to antifungal ability, ACH showed the strongest phytotoxic activity against weeds and crops, followed by HCH and CH. ACH caused chlorophyll content loss, leaf electrolytic leakage, and lipid peroxidation in a dose-dependent manner. Its phytotoxicity was stronger than that of glufosinate-ammonium but weaker than that of paraquat in the in vitro experiments. CH and its derivatives are well-known protein synthesis inhibitors; however, the precise differences between their mechanism of action remain undiscovered. A computational study revealed effects of CHs on the protein synthesis of Pythium ultimum (oomycetes), Magnaporthe oryzae (true fungus), and Capsicum annum (plant) and deciphered the differences in their biological activities on different targets. The binding energies and conformation stabilities of each chemical molecule correlated with their biological activities. Thus, molecular docking study supported the experimental results. This is the first comparative study to suggest the ribosomal protein alteration mechanisms of CHs in plants and fungi and to thus show how the protein inhibitory activities of the different derivatives are altered using molecular docking. The correlation of structures features of CHs in respect to bond formation with desired protein was revealed by density functional theory. Overall collective results suggested that CHs can be used as lead molecules in the development of more potent fungicides and herbicides molecules.

Distinguishing between long-term-stored and fresh chili pepper powder through fingerprinting of volatiles by headspace capillary-gas chromatography-ion mobility spectrometry.

Long-term storage of chili pepper powder results in physicochemical and microbiological changes that decrease its commercial value; these changes occur owing to fungal growth and production of off-flavor compounds. Herein, long-term-stored chili pepper powder (LSCPP) and fresh chili pepper powder (FCPP) were analyzed using internal transcribed spacer sequencing and volatile organic compound fingerprinting by headspace capillary-gas chromatography-ion mobility spectrometry. Fungal analysis detected only Xeromyces bisporus with high accuracy in all the analyzed LSCPP samples. However, the proliferation of X. bisporus on nonspecific spots complicated the distinguishing process between the two groups based solely on fungal analysis. Therefore, nine compounds (three ketones, one alcohol, two aldehydes, one ester, one furan, and one sulfur compound) obtained by autoxidation and fungal metabolism were selected as potential markers for distinguishing LSCPP and FCPP. These above-mentioned substances, which were confirmed as off-flavor species owing to "stale" odor, emitted lipid fragrance and were used to successfully distinguish LSCPP from FCPP using principal component analysis and linear discriminant analysis. PRACTICAL APPLICATION: According to the research results, it was possible to discriminate between long-term stored and fresh chili pepper powders using nine VOC markers for quality control in industry. In addition, the fungus generated from long-term storage of chili pepper powder was Xeromyces bisporus, which was confirmed to be safe for intake because it does not form secondary toxic metabolites.