Physicochemical, Microbial, and Volatile Compound Characteristics of Gochujang, Fermented Red Pepper Paste, Produced by Traditional Cottage Industries

Gochujang, fermented red pepper paste, is a grain-based Korean traditional food. The quality of gochujang produced by cottage industries is not well-documented. Thus, the present study aimed to analyze the quality of gochujang from 35 traditional cottage industries for physicochemical and microbial characteristics, along with volatile compound contents. In addition to microbial characteristics, salinity, pH, free amino nitrogen, and alcohol content were evaluated. Ethanol was detected as the predominant alcohol and 57% of tested gochujang products harbored >1% of total alcohol content, which was above the recommended level for halal products. Gochujang products contained hexadecanoic and linoleic acids predominantly and several volatile compounds belonging to the classes of alcohols, aldehydes, alkanes, nitrogen-containing compounds, and terpenes. A wide range of aerobic mesophilic bacteria (2.79–8.73 log CFU/g) and yeast counts (1.56–7.15 log CFU/g) was observed. Five distinct yeast species were identified, including Zygosaccharomyces rouxii. Eight gochujang products were found to be contaminated with Bacillus cereus (>4 log CFU/g). This study suggests that there is a need to limit B. cereus contamination in cottage industry products and reduce alcohol content to comply with halal food guidelines.


Introduction
Gochujang (fermented red pepper paste) is one of the most important grain-based traditional Korean fermented foods and is generally used as a sauce in Korean cuisines and as a seasoning in spicy foods. Gochujang has a distinguished flavor and savory taste [1]. In 2017, the total domestic and international retail market revenue of gochujang accounted for approximately USD 149.55 million and USD 31.98 million, respectively. Gochujang products are exported to several countries, including the US, China, Japan, and Middle Eastern countries [2]. Owing to the high amount of saccharified grain starch (from rice, wheat, or barley), and powdered red hot pepper (Capsicum annuum L.), gochujang is a red and thick paste. Furthermore, significant amounts of salt, powdered meju, and potable water are used in the preparation of gochujang. Meju is a naturally fermented soybean, which acts as the source of microorganisms (starter culture) in the fermentation of gochujang. The mixture of these ingredients starts the fermentation and aging processes [3]. Two major types of gochujang are available in the market: a modern large-scale industrial gochujang and traditional homemade gochujang [4,5]. The modern large-scale industrial gochujang is produced in a quality-controlled environment with the use of specific starter cultures (Aspergillus and Bacillus species) in a short period of fermentation [6].
The preparation of homemade and cottage industrial gochujang relies on traditional fermentation techniques using simple equipment. The traditional homemade gochujang is produced using an extensive fermentation process with natural microorganisms. The process includes saccharification via heating of glutinous rice and malt, followed by the addition of meju, red pepper powder, and salt, depending on the desired characteristics of taste and flavor, and, finally, a fermentation stage, which can last from 1 to 2 years [7]. The microbial composition of meju can affect the quality of gochujang [7]. The traditional gochujang fermentation is influenced by several elements, including local microorganisms such as the meju microflora, and surrounding environmental factors such as weather conditions [4,5,8]. Thus, the gochujang cottage industry in different provinces generates products with diversified nutritional values and organoleptic properties [9]. Raw ingredients, process methods, microorganisms involved in the fermentation, and duration of the fermentation significantly influence the organoleptic properties of gochujang, including its aroma, taste, and texture [6,8].
To make appropriate choices and optimize the production of traditional gochujang, it is essential to investigate the physicochemical and microbial characteristics. Although various laboratory and homemade unbranded gochujang products have been previously examined [9,10], these studies have not focused on the physicochemical and microbial properties and volatile compound characteristics of indigenous-branded, traditional cottage industrial gochujang products. Generally, because of the use of traditional processing technologies adopted by cottage industries, the interbatch quality of gochujang remains unvaried [10]. Hence, consumers are highly interested in indigenous branded traditional gochujang products owing to the consistently outstanding quality. These traditional cottage industries are operated with minimal capital, and thus lack a quality control department for the analyses of gochujang products. Moreover, the physicochemical and microbial features of these products are not monitored by any food and health organization and, therefore, not publicly available.
A previous study detected a significant amount of different alcohol types, particularly ethanol, in gochujang during the fermentation process [11]. In addition to the basic ethanol content, some companies supplement the product with a considerable amount of ethanol during the packaging phase to prevent microbial activity. The gochujang products containing more than 1% ethanol are prohibited for trade in Muslim countries (halal markets). Moreover, the risk of contamination of traditional cottage industry gochujang products with food pathogens, particularly Bacillus cereus, remains unexplored. The quality of gochujang products produced by cottage industries has not been sufficiently examined. This study aimed to determine the physicochemical and microbial properties, alcohol content, and volatile compounds of gochujang products collected from nationwide cottage industries, and to categorize such products based on the findings of biostatistical analyses.

Instruments and Apparatus
A pH meter (Orion Star A211, Thermo Fisher Scientific, Beverly, MA, USA) and Konica Minolta Chromameter, equipped with a CR-400 model chromameter measuring head and DP-400 model data processor, were used to measure the pH and color values, respectively. GC-MS-QP2010 SE (Shimadzu Co., Kyoto, Japan) gas chromatography-mass selective detection (GC-MSD) system with SH-Stabilwax column (30 m × 0.32 mm × 0.25 µm) and Agilent 7890B and 5977B GC-MS system (Agilent, Santa Clara, CA, USA), which includes an Agilent DB-WAX 122-7062 column (60 m × 250 µm × 0.25 µm), were used for the detection of volatile compounds and alcohol content, respectively. Plastic Petri plates (SPL Life Sciences, Pocheon, Gyeonggi, Korea) were used for the microbiological analysis. Internal transcribed spacer (ITS) sequencing of isolated microbes was conducted using the ABI PRISM 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA).

Sample Collection
A total of 35 gochujang products were purchased from various cottage industries located in different provinces of the Republic of Korea, as previously reported [1]. The major ingredients of gochujang products include red pepper powder, glutinous rice powder, powdered soybeans, grain syrup, malt, salt, and water. Detailed ingredients of the purchased gochujang products were also previously reported [1].

Physicochemical Characteristics
2.3.1. Determination of pH, Salinity, Color Values, and Free Amino Nitrogen pH values of gochujang products were analyzed according to the protocol of Ramalingam et al. [12]. The salinity of gochujang was determined using the Korea Food and Drug Administration method [13]. Color values of gochujang were obtained using a chromameter. The tristimulus color analyzer was calibrated to a reference (white porcelain plate) prior to the experiment [14]. The total free amino nitrogen contents of the gochujang samples were determined using the titration method as described by the Korea Food and Drug Administration [15] and Cho et al. [16].

Determination of Total Alcohol Content
The alcohol content profiles of gochujang products were investigated using gas chromatography-mass spectrometry (GC-MS), according to the method described by Lee et al. [17] and Gil et al. [18]. Briefly, 0.5 g of a sample was mixed with 9.5 mL of dimethyl sulfoxide and stirred at 100 rpm at 40 • C for 1 h in a 20 mL closed container. The reaction solution settled before the supernatant was filtered using the Whatman syringe filter. Subsequently, the supernatant was used for the GC-MS analysis via a GC-MSD system. A temperature of 160 • C was maintained in the GC injector, and 20 µL of the sample was injected with a split ratio of 40:1. The oven temperature was programmed to start at 40 • C for 5 min, and increase 10 • C/min up to 240 • C, and then stop at (isothermal) 240 • C for 9 min. Mass spectrum analysis (70 eV, ion-source temperature 200 • C) was performed at 0.5 s scan intervals. Standard methanol, ethanol, pentanol, butanol, and propanol solutions (0.2%) were used to estimate each alcohol concentration in the gochujang samples.

Determination of Volatile Compounds
The volatile compound profiles of gochujang products were investigated using a solidphase microextraction (SPME) method, followed by GC-MS [12]. Approximately 5 g of sample was heated to 70 • C for 20 min in a closed 20 mL container. A carbowax/divinylbenzene polydimethylsiloxane SPME fiber assembly was allowed to absorb the volatile compounds within the samples for 30 min at 70 • C. Temperatures of 250 • C and 230 • C were maintained in the GC injector and MS source, respectively. A split ratio of 20:1 was used to inject the SPME fiber at a purge flow rate of 3 mL/min (with a total flow rate of 24 mL/min) at 18.5 psi. The oven temperature was programmed to start at 40 • C for 2 min, and increase at a rate of 2 • C/min up to 220 • C and 10 • C/min up to 240 • C, and then stop at 240 • C for 10 min. Mass spectrum analysis (70 eV, ion-source temperature 230 • C) was performed at 0.5 s scan intervals. Mass spectra of the unknown compounds of samples were interpreted using the data available in the National Institute of Standards and Technology MS library [19]. The molecular weights, names, and structures of volatile compounds in the samples were determined.

Microbial Profile
The standard methods of the Association of Official Analytical Chemists [20] were adopted to analyze the total number of aerobic mesophilic bacteria and B. cereus in gochujang. 3M Petrifilm, plate count agar, and MYP culturing medium were used according to the manufacturer's protocol to estimate the total yeast and mold (yeast/mold) [21], aerobic mesophilic bacteria, and B. cereus counts, respectively. API 50CHB and API 20E kits were used to identify B. cereus using the manufacturer's protocol. PDA was used to isolate yeast/mold. The isolated yeast/mold from gochujang was subjected to ITS sequencing analysis [12]. The analyzed sequences were aligned with the help of the sequence alignment editor software BioEdit (version 7.0.4). The data on ITS sequences of the isolated microorganisms were documented in the NCBI GenBank database using the BLAST program. Phylogenetic analysis was performed for the isolated microorganisms using the neighbor-joining method [12].

Statistical Analysis
All the experiments were performed at least in triplicate, and the values were presented as the mean ± standard deviation. Statistical analyses were performed using the SPSS software 23 (IBM, Chicago, IL, USA). One-way analysis of variance in a completely randomized design and Duncan's multiple range comparison tests were used to explore the significant differences between the samples with a 95% confidence interval at p < 0.05. The multivariate exploratory techniques of principal component analysis (PCA) were conducted to categorize the gochujang samples based on their pH, lightness, redness, yellowness, amino nitrogen content, aerobic mesophilic bacteria count, yeast/mold count, and major volatile compound profile using the XLSTAT package on Microsoft Office Excel 2016 version [1].

pH
Optimal pH is one of the prerequisite physicochemical parameters of fermented foods and is the main factor influencing the occurrence of several biochemical activities [12]. All selected gochujang products exhibited acidic pH between the ranges of 3.57 ± 0.01-4.98 ± 0.01 (Table 1). The mean pH value of gochujang was 4.44 ± 0.35. Based on the pH values, all the gochujang products were grouped into two categories: samples with pH higher than 4.6 (low-acidic food), and samples with pH below 4.6 (acidic food) (USFDA, Code of Federal Regulations) [22]. A total of 40% of the gochujang products (n = 14) showed a pH higher than 4.6 (in the range of low-acid food), whereas 60% (n = 21) presented pH values below 4.6 (acidic food). The variation in pH between the different gochujang products is probably due to the origin of different basic raw materials and the contribution of different microorganisms. A previous report showed a range of low-acidic pH (4.59 ± 0.36-4.79 ± 0.15) measured in 80 different homemade gochujang products [23]. However, Lee et al. [23] did not report pH values below 4.0 for any sample. The present investigation detected a slightly acidic pH for some samples, similar to that reported by Kim et al. [4] in several laboratory-made gochujang-meju samples. In general, the initial pH values of the gochujang products ranged from 5.5-6. These values are then reduced to the level of either low-acidic food or acidic food pH values during the fermentation process [24,25]. The decrease in the pH value is dependent on the fermentation time [26], fermenting microbes [24], environmental factors [27], and raw materials [25]. The mean pH value (4.44 ± 0.35) measured in this investigation was similar to that previously reported for other gochujang products [23][24][25][26][27][28].

Salinity
The salinity of the tested gochujang products was between 3.44 ± 0.00% and 12.68 ± 0.33%, and the mean salinity value was 6.66 ± 2.18% ( Table 1). The gochujang products were categorized based on salinity in three broad groups, group I (salinity <5%), group II (salinity, 5-10%), and group III (salinity >10%). Most of the samples (68.57%) were placed in group II, followed by groups I (20%) and III (11.43%). This was due to the initial amount of salt added during the manufacturing phase of the gochujang products at the cottage industry. In the present study, all the tested gochujang products were prepared using salt supplements between 5% and 12%, which further impacted the salinity of the final product [1]. During the gochujang fermentation process, salinity increase was also detected by Beak et al. [27], whereas a decrease in salinity was reported by Ryu et al. [24]. The water content of the raw materials and external environment humidity showed a significant influence on the salt concentration of gochujang products [4]. The salinity of gochujang products reported in previous reports [23,24,27] was consistent with mean salinity observed in the present study (6.66 ± 2.18%). Moreover, Lee et al. [23] reported that none of the gochujang samples had salinity below 5% or above 10%.

Free Amino Nitrogen Content
In the 35 gochujang products, free amino nitrogen content presented mean values of 60.33 ± 32.51 mg/100 g ( Table 1). The free amino nitrogen content in all the samples ranged from 18.69 ± 0.00 mg/100 g to 168.17 ± 16.18 mg/100 g. Gochujang products were grouped into three categories based on the free amino nitrogen content, including group I (free amino nitrogen, 0-50 mg/100 g), group II (50-100 mg/100 g), and group III (100-200 mg/100 g). A total of 45.7% of the gochujang products were assigned to groups I and II, whereas only 8.6% of samples were placed in group III. It has been reported that the fermentation process increases the amino nitrogen in the gochujang products [24,[27][28][29][30]. Similarly, the prevalence of Bacillus spp. and Zygosaccharomyces spp. has a significant correlation with an amino-type nitrogen concentration of gochujang products [24]. The difference in free amino nitrogen content in the tested gochujang products was due to the distinct initial raw material used (particularly protein-rich matter), fermentation period, and the microorganisms involved in the fermentation of gochujang [24,27]. Because the 35 gochujang products were prepared with different raw materials, including powdered soybean (a major protein substrate) [12], they had diverse free amino nitrogen content. Similarly, a previous study reported the difference in the free amino nitrogen content in various industrial gochujang products [31]. Accordingly, Kim et al. [4] reported the variation in free amino nitrogen content in homemade gochujang products prepared with four different types of meju.

Color Values
The surface color of all the gochujang products was measured using a chromameter and are presented in Table 1. Color is an essential food quality for consumer acceptability. The color of the fermented food is highly dependent on the raw material used and the composition of the final product [12]. The mean values of lightness (L*), redness (a*), and yellowness (b*), of gochujang products, were 28.11 ± 2.04, 10.04 ± 3.15, and 7.59 ± 1.53, respectively. The most influential factor responsible for the redness of the products is the red pepper powder. In the present study, Go-30 displayed the highest value for redness (17.72 ± 0.13) owing to the high percentage of red pepper (34%) during preparation, whereas Go-20 had the lowest value (4.96 ± 0.12) due to the limited amount (19%) of red pepper. A previous report revealed that the progression in the fermentation process increased the a* and L* values of gochujang products, whereas b* values were decreased [24]. In another investigation, a* and L* values decreased, and no significant changes were observed in the b* values during the 1-year fermentation of gochujang products evaluated [27]. In addition to the raw material, the variation in the color values for different gochujang products is associated with the microbial composition, which metabolizes the complex biomolecules and converts them into simple molecules responsible for a unique taste, aroma, and color. The present results, including the mean color values of the gochujang products, were consistent with those reported in previous reports [24,27,28].

Alcohol Content
Alcohols, particularly ethanol, are important volatile components of fermented foods, responsible for imparting a unique flavor and aroma [32]. A wide range (0-4.99%) was noticed in the alcohol content and proportions of the tested gochujang products. The mean total alcohol content was 1.58 ± 1.28% (Figure 1 and Supplementary Table S1). Among the tested alcohols (methanol, propanol, butanol, and pentanol), ethanol content was the highest, ranging from 0 to 4.9%. Therefore, ethanol represented the single major contributor to the total alcohol content of gochujang products (Figure 1 and Supplementary Table S1). The mean ethanol content of the gochujang products was 1.53 ± 1.23%, whereas the mean methanol content was 0.004 ± 0.005% (Figure 1). None of the products showed an excessively high amount of propanol, butanol, and pentanol, and these alcohols were detected in the range from not detected to 0.016%. Gochujang products were grouped into two categories based on the total alcohol content, including group I (0-1%) and group II (>1%) [33]. Approximately 42.85% of the samples (n = 15) were placed in group I, whereas the rest of the gochujang products (n = 20) were assigned in group II. The alcohol content in 57.15% of the tested gochujang products (n = 20) was higher than the recommended amount (1%) for halal foods [32]. Moreover, six gochujang products contained more than 3% of ethanol. In these cases, the extra ethanol was added by the manufacturing companies during the packaging of gochujang products to prevent spoilage and microbial activity. Furthermore, the basic ethanol content in gochujang products is linked with types and populations of fungi participating in the fermentation [34]. In particular, Zygosaccharomyces spp. and Saccharomyces spp. yeast isolated from gochujang products produced 1.6-3.2% of the ethanol [17], thus contributing to the higher basic level of ethanol in gochujang products. Previous data generated using the electronic nose analysis of 25 traditional gochujang products revealed the presence of 0.14-2.7% of ethanol in gochujang products, and 44% of the products (n = 11) contained more than 1% of ethanol content, thus supporting the present findings [35]. An abnormally high amount of alcohol content in gochujang products leads to alteration in taste and may cause spoilage. Moreover, ethanol content higher than 1% in food restricts their consumption in Muslim countries due to halal requirements [33].

Volatile Compounds
GC-MS analysis results revealed the presence of various volatile compounds in all tested gochujang products. The total number of compounds identified in the gochujang products ranged between 53 and 104 (Supplementary Table S2). The compound names, retention times, and percentage peak area for all gochujang products are listed in Supplementary  Table S2. The predominant compound was identified as 2,3,5,6-tetramethyl pyrazine with a peak area of 54.31% and retention time of 40.054 min in Go-19, followed by ethanol with a peak area of 49.26% and retention time of 9.138 min in Go-7.
Ethanol was found in all tested gochujang products, with a peak area percentage range of 1.17-49.26%. Among the 35 gochujang products, 17 showed ethanol as the predominant component, depicting a percentage range of the highest peak area between 7.54-49.26%. Linoleic acid ethyl ester and 2,3,5,6-tetramethyl pyrazine were detected as the predominant components in 11 gochujang products with a peak area range of 7.92-31.19%, and to a lesser extent, in four other gochujang products (Go-14, Go-16, Go-19, and Go-30) with peak area range of 2.7-54.31%. Hexadecanoic acid, an ethyl/methyl ester, was detected in all gochujang products and was either the second or third most abundant compound in 26 gochujang products with a peak area range of 6.49-25.09%. Compounds detected in the gochujang products included low quantities of acids, alcohols, aldehydes, alkanes, alkenes, benzene derivatives, carboxylic acids, cyclic and bicyclic ketones, cyclosiloxanes, esters, fatty acids, furans, hydrocarbons, nitrogen-containing compounds, phenolics, pyranones, pyrazines, sulfur-containing compounds, and terpenes.
Several compounds (such as ethanol, acetic acid, benzaldehyde, benzene acetaldehyde, hexadecanoic acid, ethyl ester, hexanoic acid, hexanol, hexyl ester, linoleic acid ethyl ester, methyl salicylate, nonanoic acid, ethyl ester, octadecanoic acid, ethyl ester, 1-propanol, octanoic acid, and ethyl ester) were previously reported in gochujang [36][37][38][39][40][41][42], thus supporting the present results. Similar to the present investigation, previous studies reported a high amount of ethanol in the commercial, traditional, and improved gochujang products generated during the fermentation process via the yeast-dependent glycolytic pathway [36,37,[43][44][45][46]. Most of the ester compounds detected in the gochujang products were ethyl esters, which render a fruity aroma to the product; fatty acid esters are produced due to the esterification of organic acids and fatty acids with the ethanol generated during the fermentation process by the yeast [37]. Several aroma-active compounds, such as acetic acid (pungent sour), 2-furanmethanol (cooked sugar), methyl salicylate (peppermint), ethanol (sweet), linalool (flower and lavender), hexanal (grass, tallow, and fat), benzaldehyde (almond and burned sugar), benzene acetaldehyde (fruity and rosy), nonanal (soapy), tetramethylpyrazine (cocoa, mocha, and milk coffee), acetic acid (sour) and ethyl hexanoate (apple peel, and fruit) were detected in the tested gochujang products [37,47]. Diversified classes of compounds with different peak areas were detected in each gochujang product due to the difference in raw materials, method of raw material processing, microbial diversity during the fermentation process, and fermentation period and conditions.

Aerobic Mesophilic Bacteria and Yeast/Mold
The tested gochujang products harbored aerobic mesophilic bacteria in the range of 2.79 ± 0.10 to 8.73 ± 0.30 log CFU/g ( Table 2). The mean value of aerobic mesophilic bacteria present in the gochujang products was 6.98 ± 1.42 log CFU/g. In addition to the aerobic mesophilic bacteria, the active presence of fungi and yeast in gochujang was previously reported [7]. The yeast/mold count in all products ranged from 1.56 ± 0.06 to 7.15 ± 0.02 log CFU/g ( Table 2). The mean value of gochujang products' yeast/mold population was 4.47 ± 1.47 log CFU/g ( Table 2). In general, during the gochujang fermentation phase, aerobic mesophilic bacterial populations increased from approximately 5 log CFU/g to 8 log CFU/g, and yeast/mold counts decreased [24,26,28]. The findings from the present study are in agreement with those from previous reports that showed similar bacterial and yeast count in different gochujang products [25,26,48]. The microbial population in the products highly depended on external environmental factors, physicochemical and microbial profiles of raw materials, and the meju used as a starter culture [24,27]. Even though changes occurred in the microbial composition, the total count of aerobic bacteria was nearly constant after 3 months of gochujang fermentation [27]. Both bacteria and yeast/mold play important roles in the final features (taste, color, and aroma) of the fermented gochujang products [24,27,37,38]. Several reasons can be identified for the variation in yeast/mold count among the gochujang products, among which the selection of meju may be the most critical. The present results are in accordance with several published reports that indicated the presence of several microorganisms in gochujang [7,48,49]. In gochujang, various bacterial species have been identified and extensively studied [7,37]. However, studies regarding the presence of yeast in gochujang are limited [7]. Thus, the present investigation focused on the isolation and identification of yeast from all gochujang products. More than 100 yeast colonies were isolated from 35 gochujang products. After microscopic examination and evaluation of colony characteristics, five distinct yeast colonies were analyzed using ITS sequencing and comparative phylogenetic analysis (Supplementary Figures S1-S5). These colonies were identified as Zygosaccharomyces rouxii, Starmerella lactis-condensi, Wikerhamomyces subpelliculosus, Pichia membranifaciens, and Cladosporium welwitschiicola (Table 2). To the best of our knowledge, P. membranifaciens, C. welwitschiicola, and W. subpelliculosus were reported in the traditional gochujang products for the first time. Zygosaccharomyces rouxii was detected as a predominant yeast in 82.85% of gochujang products (n = 29). It produces several aromatic secondary metabolites during fermentation, such as esters, aldehydes, and ketones, with leavening properties [50] that improve the quality of gochujang products [24,51,52]. Z. rouxii is the main yeast species found in the traditional gochujang products, whereas Candida and Cryptococcus species were dominant in the commercial gochujang products, supporting the present results [7,48,49]. Phylogenetic analysis displayed a minor variation between the identified Z. rouxii strains. The high occurrence of Z. rouxii in gochujang products led to its high isolation frequency in the present study (Table 2), which was in accordance with the data from Jang et al. [7].

Detection of B. cereus in Gochujang Products
The presence of pathogenic bacteria in food represents a major concern for food safety. Escherichia coli, B. cereus, Salmonella species, and Staphylococcus aureus are common foodborne pathogens responsible for significant health and economic losses. Although the acidic pH of gochujang products acts as a barrier for most of the pathogenic microbes, B. cereus can proliferate in gochujang [8]. In the present study, the presence of B. cereus was observed in eight gochujang products (22.85%) at a level higher than the safety limit (4 log CFU/g) recommended by the Korean Food and Drug Administration [53] (Table 3). Yim et al. [54] measured B. cereus counts below 4 log CFU/g in all the tested commercial gochujang products. Kim et al. [10] reported the presence of B. cereus in nine industrial and 23 homemade gochujang samples and revealed that three homemade gochujang samples contained B. cereus levels higher than the safety limit. In general, the B. cereus counts increase during the gochujang fermentation process [8,27]. The source of B. cereus in gochujang may include contaminated raw materials and cross-contamination during the fermentation process. In summary, the present investigation, supported by several other studies [8,10,52], indicated that although present in gochujang products, the B. cereus count in most products was within the safety limit, suggesting that appropriate sterilization measures were adopted during the preparation process. However, a few gochujang products showed higher B cereus counts, thus leading to concerns regarding B. cereus contamination and the need for necessary preventive measures against such contamination.

Principal Component Analysis and Hierarchical Clustering of Gochujang Products
The PCA and agglomerative hierarchical clustering analysis were performed based on the physicochemical characteristics, microbial count, alcohol content, and the distribution of major volatile components of different gochujang products (Figure 2). PC1 grouped Go-1, Go-6, Go-7, Go-12, Go-17, Go-23, Go-31, and Go-34, in the positive plane from the other samples ( Figure 2A). The gochujang samples located in the positive values of PCA1 were influenced by yeast population and alcohol content. The PC2 showed the variance and grouped Go-2, Go-5, Go-19, Go-22, Go-26, Go-28, Go-29, and Go-30 (in positive values) (Figure 2A). PCA separated and grouped the different clusters of 35 gochujang samples based on their pH, salinity, free amino nitrogen, lightness, yellowness, redness, aerobic bacterial count, yeast and mold count, methanol, ethanol, propanol, pentanol, and butanol content, and major components from GC-MS analysis (ethanol, linoleic acid, and hexadecanoic acid). The gochujang products grouped in the positive region of PCA2 were influenced by free amino nitrogen content and color values. The gochujang samples in the negative plane of PCA1 and 2 varied from other samples owing to the differences in salinity, aerobic bacterial count, methanol content, and linoleic acid composition (one of the major components detected in GC-MS analysis). The pH and hexadecanoic acid content displayed significant variance in gochujang products (Go-8, Go-10, Go-11, Go-13, Go-14, Go-15, Go-21, Go-27, and Go-33) (Figure 2A). Agglomerative hierarchical clustering analysis revealed dissimilarities between the gochujang products in two key groups ( Figure 2B). The group I consisted of five closely-related clusters with 20 gochujang samples (cluster 1 = Go-29, Go-34, Go-22, and Go-30; cluster 2 = Go-3, Go-2, Go-4, and Go-18; cluster 3 = Go-16, Go-19, Go-20 Go-21, Go-14, and Go-15; cluster 4 = Go-12, and Go-17; and cluster 5 = Go-1, Go-28, Go-5, and Go-6) ( Figure 2B). Group II also comprised five clusters with 15 gochujang products (cluster 1 = Go-7 and Go-23; cluster 2 = Go-26, Go-33, Go-27, and Go-31; cluster 3 = Go-24 and Go-32; cluster 4 = Go-10, Go-8, and Go-13; and cluster 5 = Go-25, Go-35, Go-9, and Go-11) ( Figure 2B). The gochujang products within these 10 clusters were closely related in terms of tested parameters. To the best of our knowledge, no study has categorized gochujang products based on their physicochemical and microbial features using multivariate PCA. Only a limited number of previous studies have employed PCA to represent the profiling of microbes and biogenic amines in gochujang products [1,8,27,49].

Conclusions
Diversified physiochemical and microbial profiles were detected in gochujang products collected from different provinces of the Republic of Korea. This study highlighted the presence of a variety of alcohols with a predominance of ethanol in the gochujang products. A few samples had ethanol content higher than the recommended limit for halal foods. Similarly, 22% of gochujang products were contaminated with B. cereus. The study demonstrated the variations in physicochemical, microbiological, and volatile compound characteristics of gochujang products, which may be due to the influence of raw material and fermentation conditions. The variation in the microbial profile also influenced physicochemical constituents and volatile compounds of the gochujang products. Nonetheless, an exact correlation needs to be established in future studies. The outcome of the study indicates that most of the gochujang products were free from toxicogenic microorganisms, though a few gochujang products had high ethanol content and B. cereus contamination, which needs to be addressed to satisfy the guidelines of food safety and marketability. This study also recommends regular analysis of the gochujang products prepared by the cottage industry to ensure their safety toward consumers.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11030375/s1, Figure S1: Phylogenetic tree analyses of the ITS sequences of the Zygosaccharomyces rouxii isolated from gochujang products constructed in MEGA 6 software by employing UPGMA method; Figure S2. Phylogenetic tree analyses of the ITS sequences of the Starmerella lactis-condensi isolated from gochujang products constructed in MEGA 6 software by employing UPGMA method; Figure S3. Phylogenetic tree analyses of the ITS sequences of the Wikerhamomyces subpelliculosus isolated from gochujang products constructed in MEGA 6 software by employing UPGMA method; Figure S4. Phylogenetic tree analyses of the ITS sequences of the Pichia membranifaciens isolated from gochujang products constructed in MEGA 6 software by employing UPGMA method; Figure S5. Phylogenetic tree analyses of the ITS sequences of the Cladosporium welwitschiicola isolated from gochujang products constructed in MEGA 6 software by employing UPGMA method; Table S1. Various alcohol contents in gochujang products; Table S2. Various volatile compounds found in gochujang products.