Development of novel Meju starter culture using plant extracts with reduced Bacillus cereus counts and enhanced functional properties

We developed a novel type of Meju starter culture using single and combined extracts of Allium sativum (garlic clove), Nelumbo nucifera (lotus leaves), and Ginkgo biloba (ginkgo leaves) to improve the quality and functionality of Meju-based fermented products. Meju samples fermented with plant extracts (10 mg/ml) showed phenolic contents of 11.4–31.6 mg/g (gallic acid equivalents). Samples of extracts (garlic clove, lotus leaves, ginkgo leaves and their combination) fermented with Meju strongly inhibited tyrosinase, α-glucosidase, and elastase activities by 36.43–64.34%, 45.08–48.02%, and 4.52–10.90%, respectively. Specifically, ginkgo leaves extract added to fermented Meju samples at different concentrations (1% and 10%) strongly inhibited tyrosinase, α-glucosidase, and elastase activities and exhibited a potent antibacterial effect against Bacillus cereus with a significant reduction in bacterial counts compared with the effects observed for garlic clove and lotus leaf added to Meju samples. Scanning electron microscopy revealed severe morphological alterations of the B. cereus cell wall in response to ginkgo leaf extracts. Gas chromatographic mass spectroscopic analysis of plant extract-supplemented Meju samples and control Meju samples identified 113 bioactive compounds representing 98.44–99.98% total extract. The proposed approach may be useful for the development of various fermented functional foods at traditional and commercial levels.

Plant materials and preparation of extracts. All selected plant materials, including lotus leaves, ginkgo leaves, and garlic cloves were collected locally in Gyeongsan City, Korea, after which they were washed, cut, and stored at −20 °C for further processing. For preparation of ethanolic extracts, dried plant materials (100 g) were extracted with a 20-fold volume of 70% ethanol for 3 h at 65 °C. The extracts were filtered through Whatman No. 2 filter paper (Advantec; Tokyo, Japan). Filtrates of the ethanolic extracts were concentrated using a vacuum evaporator, freeze-dried, and stored at −20 °C for further use.  Table 1. To produce Meju, soybeans (as per the required amount in a Meju product) were steamed in a suitable pot and crushed, after which the ethanolic extract of each plant species (lotus leaves, ginkgo leaves, and garlic cloves) and its mixture (1:1:1 ratio) were mixed at 1% (w/w) and 10% (w/w) concentrations in a square stainless-steel plate. All the mixtures were molded into a brick shape, hung with rice straw, and allowed to ferment naturally for 60 days. Traditional Meju fermented without ethanolic extracts of medicinal plants served as a control. All Meju samples were prepared in triplicate ( Figure S1).

Physicochemical properties of plant extract-supplemented Meju samples. The physicochemical
(moisture, ash, and salt contents) or organoleptic properties (sensory effects) of the plant extract-supplemented Meju samples were determined by the methods of the Association of Official Analytical Chemists 21 (AOAC, 2006). For pH measurement of each plant extract-supplemented Meju product, the samples were diluted 10-fold with distilled water and then homogenized, followed by filtration through Whatman No. 2 filter paper (Advantec, Tokyo, Japan). The pH level was measured using a pH meter (Orion 35 star pH Benchtop, Thermo Electron Corporation; Beverly, MA, USA). Extraction procedure. Meju samples (10 g each) prepared using the ethanolic plant extracts were subjected to reflux extraction with 200 ml of distilled water for 3 h at 70 °C, followed by filtration through Whatman No. 2 filter paper. The residue was again extracted twice with an equal volume of water for 3 h. The filtrate was then freeze-dried. The yields of extracts of Meju samples were in the range of 22.1-25.25%.
Determination of total phenolic content. The total phenolic content of each Meju sample prepared from the ethanolic plant extracts was determined using Folin-Ciocalteu's reagent with gallic acid as a standard phenolic compound 22 . Briefly, 20 µl of each diluted Meju sample extract (1 mg/ml) was added to 100 µl of Folin-Ciocalteu's reagent. After 3 min, 80 ml of a 10% aqueous sodium carbonate solution was added to the mixture. The solution was allowed to stand for 1 h at room temperature (RT), and absorbance of the resulting blue mixture was measured at 765 nm against a blank containing only the extraction solvent (200 µl). Total phenolic content was calculated as gallic acid equivalents (GAE) via the calibration curve constructed using the gallic acid standard solution and expressed in mg GAE/g dry mass.
Determination of total flavonoid content. The total flavonoid content of each Meju sample prepared from ethanolic plant extracts was determined by the colorimetric method 23 . Briefly, 100 μl of each extract or standard reagent and 400 μl of ethanol were mixed with 500 μl of a 2% AlCl 3 solution (diluted in distilled water). After 1 h of incubation at RT, absorbance was measured at 430 nm. Quercetin served as a reference compound to generate a standard curve, and results were expressed in milligrams of quercetin equivalents (mg QE/g dry mass).

Determination of tyrosinase inhibitory activity of Meju samples. Tyrosinase inhibitory activity of
Meju samples supplemented with plant extracts was determined by a method described previously 24  separately added to a reaction mixture containing 40 µl of a 10 mM DOPA solution, 50 µl of 0.175 M sodium phosphate buffer (pH 6.8), and 40 µl of a mushroom tyrosinase solution (110 U/ml). The reaction mixture was allowed to incubate at 37 °C for 2 min, after which the amount of dopachrome produced was measured at 475 nm on a microtiter plate reader. Kojic acid (62.5, 125, 250, 500 and 1000 µg/ml) was used as a standard compound for a positive control. The percentage of tyrosinase inhibition was calculated as follows: sample blank Determination of α-glucosidase inhibitory activity of Meju samples. α-Glucosidase inhibitory activity of Meju samples supplemented with plant extracts was evaluated according to a chromogenic method 25 , with minor modifications. Briefly, various concentrations (5, 10, 20, and 50 mg/ml) of Meju samples (50 µl) and 100 µl of α-glucosidase (1.0 U/ml) dissolved in 0.1 M phosphate buffer (pH 6.9) were mixed in a 96-well microplate and incubated at 25 °C for 10 min. After pre-incubation, 50 µl of p-nitrophenyl-α-D-glucopyranoside (5 mM) in 0.1 M phosphate buffer (pH 6.9) was added into each well as a substrate solution. The reaction mixture was incubated at 25 °C for 5 min. Absorbance was recorded using a microtiter plate reader at 405 nm before and after incubation with a p-nitrophenyl-α-D-glucopyranoside solution and compared with that of a control containing only 50 µl of the buffer. Acarbose at various concentrations (31.3, 62.5, 125, 250, 500, 1000, and 2000 µg/ ml) was used as a standard compound. Each experiment was conducted in triplicate, and the enzyme inhibitory rates of the samples were calculated as follows:  (5,10,20, and 50 mg/ml) were mixed and incubated for 15 min. Then, 10 μl of 2.5 U/ml elastase (optimum reactivity of the enzyme) was added, after which the reaction mixture was incubated for another 15 min; the absorbance was then measured at 410 nm. Ursolic acid at various concentrations (100, 250, 500, and 1000 and 10000 µg/ml) served as a standard compound. The elastase inhibitory rate was calculated as follows: Inhibitory spectrum of the tested plant extracts against B. cereus. Growth and preparation of the bacterial strain. B. cereus was grown in NB at 37 °C for 18-24 h. After proper bacterial growth, the bacterial culture was diluted with peptone water to adjust it to the proper concentration (10 7 CFU/ml) and was exposed to the extract solution (1% and 10%) of A. sativum, G. biloba, or N. nucifera. Prior to scanning electron microscopy (SEM) analysis, we also performed other inhibitory assays to confirm the inhibitory effects of the plant extracts. The details of the method are described in Supplementary Information Section 1.
SEM analysis. SEM was conducted to determine the effects of all tested plant extracts on the morphology of the B. cereus under study at the minimum inhibitory concentration (MIC). Briefly, the bacterial cells (10 7 CFU/ml) of the control and treatment groups were washed with 0.05 M phosphate buffer (pH 7.0), followed by centrifugation (4000 × g, 10 min), and the cells were first fixed in 2.5% (w/v) glutaraldehyde at room temperature for 2 h. Secondary fixation was conducted in a 1% (v/v) arsenic acid solution at 4 °C overnight. Sample groups were dehydrated by increasing the concentration of ethanol and freeze-dried. Control samples were prepared without the addition of plant extract samples. To examine morphological changes, a published SEM protocol 29 was modified. Finally, each bacterial sample was sputter-coated with gold in an ion coater for 2 min, followed by examination under a scanning electron microscope (Hitachi; Hitachi City, Japan).

GC-MS profile of bioactive compounds in Meju samples. Extraction of bioactive compounds from
Meju. For GC-MS analysis, different Meju samples were subjected to extraction as suggested by Namgung et al. 30 .
Briefly, Meju samples (5 g each) were extracted using 50 ml of 80% methanol in water (v/v) at 70 °C for 30 min, followed by filtration through Whatman No. 2 filter paper. The residue was again extracted twice with the same volume of 80% methanol for 30 min, followed by filtration. The filtrate was then concentrated under reduced pressure and diluted up to a volume of 10 ml with 80% methanol, followed by centrifugation (10,000 rpm; 10 min). Next, the transparent upper layer of each extract (1 ml) was transferred into a glass vial, and methanol was evaporated under a constant stream of nitrogen at 40 °C, followed by derivatization.
Derivatization of Meju sample extracts. For derivatization, 50 µl of the methoxyamine hydrochloride reagent (50 mg dissolved in 1 ml of pyridine) was added to each methanol-extracted Meju sample in an extraction vial; the components were mixed and kept at 40 °C for 60 min. Then, 100 µl of the N-methyl-N-(trimethylsilyl)-trifluor oacetamide reagent was added into each vial, followed by incubation at 40 °C for 45 min 31 . Finally, the derivatized samples were filtered using syringe filters (0.45 µM) and kept in GC vials. Bioactive compounds in Meju samples were identified on a GC-MS system (Agilent; Santa Clara, CA, USA).

GC-MS conditions and analysis.
Chromatography was carried out on a Hewlett-Packard (Palo Alto, CA, USA) fused silica column DB-5 MS UI (30 m length, 0.25 mm inner diameter, and 0.25 mm thickness). The GC-MS conditions were reported elsewhere 31 . The relative proportions of the extract constituents were expressed as percentages by peak area normalization. Extract components were identified based on GC retention time relative to computer matching of mass spectra using Wiley and National Institute of Standards and Technology libraries for the GC-MS system.
Statistical analysis. The data were expressed as the mean ± standard deviation of three independent experiments and subjected to one-way analysis of variance and Student's t test. Data with P values < 0.05 were considered statistically significant. IC 50 values (concentration required for 50% inhibition) were calculated using the formula Y = 100 × A1/(X + A1), where A1 = IC 50 , Y = response, X = inhibitory concentration (linear regression analysis).

Data availability.
The authors declare that all the other data supporting the finding of this study are available within the article and from the corresponding author on reasonable request.

Results and Discussion
Production of Meju fermented with plant extracts. All Meju samples were prepared in two different sets to determine the variability of the results, and the microflora in each lot was analyzed. As presented in Figure S1, the presence of natural microflora (fungal growth) in Meju samples fermented for 60 days in a natural environment with and without plant extracts confirmed the fermentation of Meju samples (control as well as plant extract-supplemented samples).

Physico chemical properties of Meju samples.
To assess the acceptability of the plant extract-supplemented Meju products, physico chemical properties were analyzed by the standard AOAC methods 21 .
Ash content indicates the amount of mineral salts present in the diet 32  Total phenolic and flavonoid contents. There is great interest in natural phenolic and flavonoid antioxidants because of their presence in edible plants, their health benefits, and their possible use as natural food preservatives 34 . Raw materials such as soybeans and plants are rich in various bioactive phytochemicals (phenolics and flavonoids). Therefore, in the present study, we analyzed the total phenolic and flavonoid contents of Meju samples supplemented with plant extracts and compared the values with those of traditional Meju samples ( Table 2). The total phenolic content of Meju samples containing 10% lotus leaf extract (LOM10) showed the highest value, 31.6 ± 0.51 mg GAE/g. On the other hand, the flavonoid contents of Meju samples supplemented with 10% plant extracts (GAM10, LOM10, GIM10, and MIM10) were 7.13 ± 0.07, 23.75 ± 0.62, 7.90 ± 0.14, and 11.69 ± 0.19 mg QE/g, respectively, and were higher than the flavonoid content of the control Meju (7.47 ± 0.28 mg QE/g) prepared without any plant extracts (Table 2). However, some researchers have reported that the flavonoid content of fermented soybean products may vary owing to differences in the raw materials used during fermentation 35,36 . Lin et al. 37 suggested that the starter culture or microflora can affect the antioxidant activity of soybeans after fermentation. Nam et al. 38 reported that flavonoid content can be significantly affected by the soybean cultivar and fermentation period. These results suggest that higher antioxidant activity may be due to the presence of phenolic and flavonoid compounds in Meju samples fermented with various plant extracts. Hence, extracts with a high polyphenolic content likely have strong antioxidant activity.
Scientific RepoRts | 7: 11409 | DOI:10.1038/s41598-017-09551-0 Tyrosinase inhibitory activity. Tyrosinase is a major enzyme of the melanin synthesis pathway in melanocytes. Because melanin causes dark spots and freckles on the skin 39, 40 , inhibition of tyrosinase could be an important strategy for blocking melanogenesis 40 . Commercial tyrosinase inhibitors such as kojic acid, arbutin, ascorbic acid derivatives, retinoic acid, and azelaic acid are used as ingredients in cosmetics to prevent hyperpigmentation owing to their skin-whitening efficacy 39 Table 2). The majority of natural tyrosinase inhibitors derived from functional food products (food/formulations) consist of phenolic or flavonoid components 39 . In addition, the tyrosinase inhibitory activity may depend on the hydroxyl groups of phenolic compounds, which form hydrogen bonds with the enzyme's active site, thereby causing steric hindrance, conformational changes, and ultimately suppression of enzymatic activity 41 . Thus, the phenolics present in our Meju samples may play a major role in inhibiting tyrosinase activity. Polyphenols may also be used as depigmentation agents because of their structural similarity to tyrosine, a substrate of tyrosinase 41 . In addition, antioxidants inhibit pigmentation by various mechanisms, including scavenging of ROS and reactive nitrogen species as well as reduction of o-quinones or other intermediates of melanin biosynthesis, thus delaying oxidative polymerization 42 . Therefore, polyphenolic compounds are partially responsible for the efficacy

Elastase inhibitory activity. Elastin is a major component of skin connective tissue and plays an important
role in maintaining skin elasticity 39 . Elastin also participates in the formation of a network with collagenous fibers under the epidermis 45 . Elastase hydrolyzes peripheral and structural proteins in dermal connective tissue and has a strong ability to degrade elastin 45 . Because decomposition of elastin results from the activation of elastase caused by UV irradiation or ROS formation, inhibition of elastase activity could be a therapeutic target for protection against elastin-induced skin aging 46 . The active ingredient responsible for this inhibitory activity is believed to be phenolics and flavonoids present in plant extracts 47 .
The elastase inhibitory activities of Meju samples supplemented with plant extracts individually or in combination and the activity of a Meju sample without plant extracts are shown in Fig. 2. In this assay, Meju samples supplemented with extracts individually and in combination yielded optimal results at 20 mg/ml. Notably, all the tested Meju samples (20 mg/ml) supplemented with plant extracts of garlic cloves, lotus leaves, and ginkgo leaves individually or in combination (1:1:1) exerted elastase inhibitory activities of 9.90 ± 1.92%, 8.74 ± 1.07%, 3.01 ± 1.35%, and 5.52 ± 1.01%, respectively, although their values were lower than the activity of the control Meju sample (11.18 ± 1.51%; Fig. 2). In this assay, the IC 50 values of all the experimental and control Meju samples were 199.70-1364.86 mg/ml and 171.86 mg/ml, respectively. On the other hand, the IC 50 value of the positive control (ursolic acid) for elastase inhibition was 0.85 mg/ml ( Table 2). Although the Meju samples supplemented with plant extracts did not show significant elastase inhibitory activities for possible skin improvement, they showed positive results regarding melanin-reducing (tyrosinase inhibitory) activity.
In vitro studies on both purified elastases and cultured fibroblasts showed that soybean extracts can affect the extracellular matrix and inhibit enzymatic activities of several elastases 48 . Based on these studies, it can be inferred that all our Meju samples fermented with plant extracts as well as the control Meju sample may have beneficial effects on human skin and certain dermatological disorders because of the enhanced production of bioactive compounds (phenolics and flavonoids) during fermentation. The increasing use of phenolic and flavonoid compounds in the cosmetic industry could be attributed to the compounds' health benefits, including antioxidant properties and skin-improving effects 49 . These findings suggest that the consumption of Meju supplemented with plant extracts may have positive effects and be useful for the treatment of skin pigmentation. α-Glucosidase inhibitory activity. The application of Meju samples fermented with herbal extracts for treating diabetes remains largely unexplored; this situation has prompted some scientists to develop novel agents with inhibitory effects on intestinal glucosidases. The in vitro α-glucosidase inhibitory activities of Meju samples are summarized in Table 2 and Fig. 3. The α-glucosidase inhibitory activities of all the tested Meju samples fermented with extracts of garlic cloves, lotus leaves, and ginkgo leaves individually or in combination (1:1:1) were determined using p-nitrophenyl-α-D-glucopyranoside as a substrate and compared with the activity of the control Meju sample (without addition of plant extracts; Table 2). Acarbose served as a standard compound. In this assay, the percentages of α-glucosidase inhibition shown by Meju samples fermented with individual plant extracts were similar to that of Meju samples fermented with a combination of extracts (1:1:1), indicating a synergistic or additive interaction between the plant extracts and Meju samples. The control Meju sample showed  Table 2). The control Meju sample showed a higher IC 50 value (10.48 mg/ml) than did the Meju sample supplemented with lotus leaf extract (LOM1 and LOM10: 5.83 mg/ml and 4.87 mg/ml, respectively; Table 2). On the other hand, the IC 50 value of the standard compound (acarbose) for α-glucosidase inhibition was 0.70 mg/ml ( Table 2). In addition, all the tested Meju samples (20 mg/ml) supplemented with a 1% plant extract of garlic cloves, lotus leaves, and ginkgo leaves individually or in combination exerted considerable α-glucosidase inhibitory activities: 68.66% ± 4.47%, 72.12% ± 9.71%, 77.94 ± 2.04%, and 63.76 ± 4.60%, respectively. In contrast, this inhibitory activity was slightly lower for the control Meju sample (60.07 ± 6.53%; Fig. 3). The inhibitory activities of Meju samples fermented with plant extracts against α-glucosidase may be due to the increased glycoside content of soybeans caused by either fermentation or the addition of plant extracts. Glycosides consist of sugars that may be structurally similar to the carbohydrate substrate of α-glucosidase 50 . Meju samples fermented with plant extracts were found to have lower IC 50 values than the control Meju sample because their active chemical compounds did not undergo fractionation and may synergistically inhibit α-glucosidase 51 . Despite several studies on Doenjang samples 31,35,43,44,51 with Meju as a fermentation starter, there has been no investigation of enzyme-based α-glucosidase inhibition in normal Meju samples. In addition, there are various scientific reports on the health benefits of garlic cloves, lotus leaves, and ginkgo leaves, including anti-obesity and anti-diabetic effects 52,53 , suggesting that Meju samples fermented with these plant extracts could be used to develop functional foods for the prevention of diabetes with enhanced biological activities.

Reduction in B. cereus counts in Meju samples fermented with plant extracts.
In this assay, all the selected plant extracts derived from garlic cloves, lotus leaves, and ginkgo leaves individually or in combination (1:1:1) were used to prepare Meju during fermentation to examine their ability to reduce B. cereus counts in samples of the soybean-based Meju starter culture. Results showed that B. cereus counts in Meju samples fermented with individual plant extracts of garlic cloves, lotus leaves, and ginkgo leaves or their combination were drastically reduced compared with those in the traditional Meju sample fermented without any plant extract. Meju samples fermented with individual plant extracts showed B. cereus counts ranging from 0 CFU/g to (1.0 ± 0.1) × 10 2 CFU/g. On the other hand, the control Meju sample showed B. cereus counts ranging from (1.7 ± 0.01) × 10 3 CFU/g to (1.8 ± 0.07) × 10 4 CFU/g (Table 3). Meju samples fermented with a higher concentration (10%) of plant extracts inhibited the growth of B. cereus better than samples fermented with a low concentration (1%) of plant extracts (Table 3). To confirm that the bacterium found in the Meju samples (1% plant extract-supplemented Meju and control Meju) was B. cereus, colonies on MYP agar were identified using the API kit, and all the analyzed colonies tested positive for B. cereus with 99.9% ID and T-index values ranging from 0.37 to 0.62. These data confirmed how closely the profile corresponds to the taxon in question relative to all other taxa in the database, and how closely the profile corresponds to the most typical set of reactions for each taxon, respectively (Table 3). Moreover, among all the Meju samples fermented with plant extracts, the Meju samples that were fermented with the 1% and 10% ginkgo leaf extract showed a remarkable reduction in B. cereus counts (Table 3). Confirmatory tests for counted B. cereus cells were also performed by molecular identification. Results showed that all the colonies suspected of containing B. cereus tested positive for B. cereus with 100% similarity (data not shown).
According to the abovementioned results, the addition of 10% ethanolic extracts of selected plants individually or in combination may be a feasible approach to controlling the growth of food-borne B. cereus in starter culture Meju samples. Lim and Lee 13 observed a growth reduction of B. cereus in Meju samples prepared from licorice extracts at different concentrations. Although various methods for inactivating B. cereus have been developed, including ethanol, sodium chloride, and microwave methods 13,54 , the application of plant extracts of natural origin to Meju samples appears to be the most effective method for controlling the growth of hazardous pathogens such as B. cereus. In addition, Lim and Lee 13 produced licorice extracts to enhance Meju samples with respect to sensory attributes and consumer acceptability. In the present work, fermented soybean paste samples were approved by performing sensory examinations and other consumer acceptability tests, which were not elaborated when licorice extracts were incorporated into Meju samples. Furthermore, the Korean population is well aware of the use of a few of these plant extracts (garlic cloves and lotus leaves) in different types of fermented soybean products. These findings suggest that selected plant extracts derived from garlic cloves, lotus leaves, and ginkgo leaves individually or in combination can be utilized in conjunction with other conventional food safety measures to inhibit the growth of B. cereus in Meju samples.

Confirmation of inhibitory effects of plant extracts against B. cereus. Physical and morphological
alterations may induce deterioration of the cell wall surface of bacterial pathogens upon treatment with a suitable antimicrobial agent. Hence, SEM analysis was carried out to visualize the effects of these plant extracts individually and in combination on the morphology of B. cereus cells relative to a control group (Fig. 4). Ethanolic extracts of lotus leaves and ginkgo leaves revealed possible inhibitory effects, as confirmed by severe morphological alteration of the cell wall of B. cereus, leading to cell disruption and lysis ( Fig. 4B and C). Furthermore, B. cereus cells treated with plant extracts showed severely damaged cell morphology, including disruption of the cell membrane, abnormal cell breaking, and swelling. In contrast, control cells of the pathogen tested (in the absence of any plant extract) showed regular and smooth surfaces (Fig. 4A). In our preliminary study, the in vitro inhibitory activities of ethanolic extracts of garlic cloves, lotus leaves, ginkgo leaves added individually and in combination (1:1:1) against B. cereus were qualitatively and quantitatively assessed by the presence of inhibition zones and MIC values (Supplementary Section 1). Results showed that extracts derived from ginkgo leaves, lotus leaves, and combinations thereof had possible inhibitory effects on B. cereus (data not shown). Several researchers have also demonstrated the anti-bacterial effects of garlic cloves, lotus leaves, and ginkgo leaves against a variety of food-borne pathogenic bacterial strains, including B. cereus 55,56 .
The literature suggests that the active ingredients of plant extracts such as phenolics and flavonoids may bind to the cell surface and penetrate target sites such as the plasma membrane containing membrane-bound enzymes, disrupting the cell wall structure 57 . Thus, among all the tested plant extracts and combinations thereof, the ethanolic extract of ginkgo leaves showed the strongest bactericidal activity, as indicated by the significant reduction in microbial counts and complete inhibition of B. cereus growth. Based on these results, selected plant extracts can be considered promising anti-microbials for improving food safety by controlling B. cereus counts in food products.
In our previous study, we evaluated the fungal microflora in Meju samples fermented with single and/or multiple plant extracts, including lotus leaves, ginkgo leaves, and garlic cloves, at different concentrations (1%, 10%, and a mixture of these extracts in a ratio of 1:1:1). Meju samples fermented with individual and/or multiple plant extracts showed the presence of various non-hazardous fungal strains, including Aspergillus species, Mucor species, Paecilomyces species, and Penicillium chrysogenum. In contrast, the control Meju samples fermented without plant extracts revealed the presence of other fungal microflora such as Aspergillus ruber 58 , which is known to produce aflatoxin B1 and ochratoxin A 59 . The findings of the present study confirm that the fermentation of Meju with plant extracts may strongly affect the fungal microflora by eliminating hazardous fungal pathogens through the suppressive effects of the extracts against toxin-producing fungal pathogens, improving the quality of Meju.
In addition, we analyzed functional properties of selected plant extracts. The literature and our experimental data show that these plant extracts and their bioactive compounds have various known pharmacological effects 18,19 . As a reference, there are many other plant components that are directly related to several other functional activities, including anti-microbial activities 18 . Additionally, similar studies have demonstrated the direct and indirect inhibitory effects of several plant extracts and their active ingredients on certain biological activities 60,61 .  Table 3. Bacillus cereus counts from Meju samples added with and without plant extracts. 1 Mannitol-egg yolkpolymyxin agar plate; 2 Analytical profile index. *Percent similarity; that is, how closely the profile corresponds to the taxon relative to all other taxon in the database. **T-index; that is, how closely the profile corresponds to the most typical set of reactions for each taxon.

GC-MS profile of bioactive compounds in Meju samples. GC-MS is an analytical method that com-
bines the features of gas-liquid chromatography and mass spectrometry to identify different substances in a variety of samples. GC-MS analysis of plant extract-supplemented Meju samples and a control Meju sample resulted in the identification of 113 compounds representing 98.44-99.98% of the total extract. All tested Meju samples yielded compounds largely composed of alcohols, sugars, amides, furanones, phenolics, terpenoids, steroids, alkaloids, and flavanones, as well as hydrazine and imidazole derivatives and other phytochemicals. It was noted in this study that the percentage of compounds increased in all the tested Meju samples as the concentration of plant extracts increased from 1% to 10% (Table S1). A detailed chemical profile of various plant extract-supplemented Meju samples and the control Meju sample (with no added plant extracts) is presented in Table S1. Docosenamide (Table S1), an amide compound, was present in all the tested Meju samples. After comparing with control Meju samples, the 1% plant extract-supplemented samples showed relative areas ranging from 0.12% to 0.14%, whereas 10% plant extract-supplemented Meju samples yielded a higher range (0.27-0.89%) of the relative area. Ghazali et al. 62 reported the presence of docosenamide in the root extract of Ixora coccinea, which showed anti-microbial effects. Kitaoka et al. 63 isolated and purified laminaribiose from Euglena gracilis, a disaccharide important in the field of agriculture as an antiseptic. Laminaribiose (Table S1) was also present in Meju samples supplemented with garlic cloves, lotus leaves, or ginkgo leaves as well as in mixed Meju samples, but it was absent in the control Meju sample. It was found that in the 10% plant extract-supplemented Meju samples, the relative peak area was slightly larger (0.19-0.35%) than that for the 1% plant extract-supplemented Meju samples (0.07-0.18%). Another key compound that was present in all the tested Meju samples is erythritol, a four-carbon sugar alcohol (polyol). Generally recognized as safe, erythritol is used in the food industry as a low-calorie sweetener 64 . The alkaloid N-methylasimilobine, which was present only in 1% and 10% lotus leaf extract-supplemented Meju samples and in 1% and 10% mixed extract-supplemented Meju samples, has been reported to possess acetylcholinesterase inhibitory activity 65 . In our previous study 18 , we reported the presence of N-methylasimilobine in the methanol extract of lotus leaves, indicating the potential therapeutic usefulness of this extract.
In addition, control and plant extract-supplemented Meju samples contained an aromatic acid component, azaazoniaboratine (Table S1), which has been found to exert an anti-cancer effect 66 . On the other hand, furanone and its various derivatives possessing antioxidant and anti-inflammatory activities 67 were also present in the Meju samples analyzed, indicating the medicinal utility of the Meju samples under study. Among furan derivatives, the furan ring is a constituent of several important natural products, including fructofuranose, psicofuranose, sorbofuranose, allofuranose, and many other natural terpenoids.
Other volatile organic acids such as lactic acid, acetic acid, propanoic acid, citric acid, acrylic acid, palmitic acid, and butenedioic acid were also present in all the tested Meju samples and can provide an important contribution to the flavor characteristics of various foods while imparting antioxidant and other pharmacologically important activities. These results are in strong agreement with the findings of Tang et al. 68 . Propionic acid is a naturally occurring carboxylic acid that inhibits the growth of molds and some bacteria when added to food at concentrations between 0.1% and 1% 69 ; the acid was also detected in our Meju samples. Moreover, anthraquinone, which has been reported to possess strong anti-microbial properties 70 , was found in abundance in garlic and ginkgo leaf extract-supplemented Meju samples and in mixed-extract-supplemented Meju samples, suggesting that anthraquinone alone or in combination with other bioactive compounds of Meju samples may be responsible for the observed anti-microbial effect. In this study, GC-MS analysis of different Meju samples detected the presence of common bioactive compounds and specific compounds because different plant extracts were used for Meju production.

Conclusions
The purpose of this study was to develop a new system for producing novel types of traditional Korean Meju products by incorporating various plant extracts with significant biological effects during fermentation. GC-MS analysis of various plant extract-supplemented Meju samples identified various bioactive compounds such as organic acids, amino acids, fatty acids, terpenes, phenolics, sterols, alkaloids, flavonoids, and sugars, as well as furans and their derivatives. Moreover, the percentage of various bioactive compounds found in plant extract-supplemented Meju samples gradually increased with the concentration of plant extracts during Meju fermentation. Fermented soy products such as Meju prepared from selected plant extracts derived from garlic cloves, lotus leaves, and ginkgo leaves individually or in combination at different concentrations (1% and 10%) exerted strong inhibitory effects against the growth of B. cereus. Moreover, the fermented Meju samples incorporated with ginkgo leaf extract (10%) showed considerable tyrosinase and α-glucosidase inhibitory effect compared with traditional Meju samples fermented without plant extracts. These findings reinforce the notion that the various biological and functional properties observed for Meju samples incorporated with ginkgo leaf extract may be attributed to various biologically active polyphenolic compounds, which may act either individually or synergistically. Based on the abovementioned findings, it can be concluded that these plant extracts can serve as natural additives during the production of functional Meju products with acceptable attributes.