(Gochujang) during Fermentation - Springer Link

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Oct 31, 2013 - Jeong-Yong Cho, Hyoung Jae Lee, Heung-Chule Shin, Jeong-Mi Lee, Keun-Hyung Park, and Jae-Hak Moon. Received: 24 January 2013 ...
Food Sci. Biotechnol. 22(5): 1245-1252 (2013) DOI 10.1007/s10068-013-0208-4

RESEARCH ARTICLE

Behavior of Flavonoid Glycosides Contained in Korean Red Pepper Paste (Gochujang) during Fermentation: Participation of a β-Glucosidase Inhibitor Jeong-Yong Cho, Hyoung Jae Lee, Heung-Chule Shin, Jeong-Mi Lee, Keun-Hyung Park, and Jae-Hak Moon

Received: 24 January 2013 / Revised: 20 March 2013 / Accepted: 28 March 2013 / Published Online: 31 October 2013 © KoSFoST and Springer 2013

Abstract Gochujang is a red pepper-soybean paste and a popular Korean fermented food. The aim of this study was to investigate the behavior of flavonoids and activity of βglucosidase during fermentation of gochujang prepared using Aspergillus oryzae. The content of flavonoid glycosides (genistin, daidzin, apigenin 7-O-β-D-glucopyranoside, and quercetin 3-O-α-L-rhamnopyranoside) and their aglycones did not change significantly during preparation of gochujang. However, β-glucosidase was produced by A. oryzae. The polar layer of the gochujang methanol (MeOH) extract more strongly inhibited the β-glucosidase activity of the crude enzyme extracted from gochujang than that of the nonpolar layer. The red pepper extract and high salt solution of the gochujang ingredients showed high βglucosidase inhibition activity. These results indicate that flavonoid glycosides were not hydrolyzed by the βglucosidase produced by A. oryzae during gochujang fermentation, which may have been due to the salt and hydrophilic compound(s) present in red pepper as main ingredients of gochujang. Keywords: gochujang, flavonoid glycoside, red pepper, βglucosidase inhibitor, Aspergillus oryzae

Jeong-Yong Cho, Hyoung Jae Lee, Heung-Chule Shin, Keun-Hyung Park, Jae-Hak Moon () Department of Food Science and Technology, and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea. Tel: +82-62-530-2141; Fax: +82-62-530-2149 E-mail: [email protected] Jeong-Mi Lee Daesang R & D Center, Icheon 467-813, Korea Heung-Chule Shin Present address: HiteJinro Co., Ltd., Jeonbuk, Korea

Introduction Gochujang is a red pepper-soybean paste and a representative Korean fermented food along with soybean paste (doenjang) and soy sauce (ganjang) (1). Gochujang has been extensively used as a sauce in Korean cooking and spicy seasonings. Gochujang is prepared by fermentation after mixing the primary ingredients, including red hot pepper powder, waxy rice flour, and meju (fermented soybean). The starter for gochujang fermentation is the natural microflora in meju or appended pure strains such as Aspergillus and Bacillus sp. (2,3). Gochujang can have a sweet, savory, and/or pungent taste depending on its primary ingredients and kinds of microorganisms. In particular, Aspergillus oryzae is generally used to mass produce gochujang in quality controlled factories. The major ingredients of gochujang are red pepper and meju. Therefore, various beneficial compounds such as capsaicin derivatives in the red pepper and isoflavones in meju may contribute to the biological effects of gochujang (4,5). Several studies have shown that gochujang produces various biological effects, including antiobesity (6,7), antitumor (8), reduction of insulin resistance (9), and anticancer (10) activities. In general, chemical analyses of the constituents of fermented foods offer important information concerning the standardization, quality control, and physiological activity of fermented foods. However, the bioactive compounds contained in gochujang have not been investigated. In our previous studies, we isolated 3 organic acids, 8 phenolic acids and their derivatives, 5 flavonoid glycosides, and 2 acyclic diterpene glycosides from gochujang methanol (MeOH) extracts (11,12). Interestingly, of the isolated compounds, the flavonoids were isolated in glycosidic forms, such as daidzin (D7G), genistin (G7G), apigenin 7-O-β-D-apiofuranosyl (12)-β-D-

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glucopyranoside, apigenin 7-O-β-D-glucopyranoside (A7G), and quercetin 3-O-α-L-rhamnopyranoside (Q3R). D7G and G7G are representative soybean isoflavonoid glucosides (13), and therefore, it is thought that these compounds may be derived from meju in gochujang, which is prepared by soybean fermentation. Other flavonoid glycosides are assumed to be from red pepper, as their presence in pepper has already been reported (14,15). Most flavonoids are found in their glycosidic forms in nature (13,16). Flavonoids are well known to exert health beneficial effects such as antioxidant, anti-inflammatory, and anticancer activities (17,18). In addition, flavonoid aglycones have been sometimes reported to show relatively higher biological activities than their glycosides (16,19,20). Many studies have examined the hydrolysis and bioconversion of flavonoid glycosides during the manufacturing of fermented foods (21,22). However, in our previous study (11), the flavonoids contained in gochujang after fermentation were in glycosidic forms, even though gochujang is made by fermentation. These findings suggest that flavonoid glycosides may be not hydrolyzed during gochujang manufacturing. Many studies have examined the effects of additives on the taste (23) and flavor components (24,25) of gochujang and have screened different microflora (26) with the goal of improving gochujang quality (2,3). However, the activity of bioactive compounds in gochujang during fermentation has not yet been systematically evaluated. In particular, the presence or absence of hydrolysis of flavonoid glycosides during gochujang manufacturing is unknown. In this study, the behavior of flavonoids and their glycosides, as well as β-glucosidase activity during manufacturing of gochujang fermented with A. oryzae was examined. We also investigated the origin of a βglucosidase inhibitor in the main ingredients of gochujang.

Materials and Methods Materials Preparation of the gochujang was carried out according to the mass-production method in small scale (23), with slight modifications. Gochujang materials including red pepper (Capsicum annuum L.) powder, wheat flour, and domestic solar sea salt were obtained from a local market in Sunchang. Soybean and non-glutinous rice were purchased at the same local market to prepare the meju and koji. Non-glutinous rice and soybean were soaked for 24 h at room temperature, drained, and steamed for 30 min at 121oC, respectively. After cooling, the steamed non-glutinous rice was inoculated with a 5%(v/w) spore suspension of A. oryzae and incubated for 2 days at 30oC in an incubator (DS-31F; Dasol Scietific Co., Ltd., Hwaseong, Korea) to obtain rice koji. The steamed soybean was pounded and

Cho et al.

inoculated with 0.3%(w/w) rice koji prepared with A. oryzae to prepare the meju. This mixture was fermented for 45 days at 30oC without a mold step to give meju. The meju was ground finely to pass through a 425-µm sieve. Wheat flour was sprayed with warm water and then steamed for 30 min at 121oC. Gochujang was prepared with red pepper powder (17.8%), steamed wheat flour (32.3%), meju (1.8%), koji (0.3%), salt (10.1%), and water (37.7%). The mixture of ingredients was fermented at 30oC for 30 days in an incubator (DS-31F; Dasol Scietific Co., Ltd.). The samples were collected at intervals of 0, 6, 12, 18, 24, and 30 days during the fermentation. The samples obtained during gochujang manufacturing were immediately stored at 70oC until use for chemical profiling of flavonoids and their glycosides and to determine βglucosidase activity. Chemicals Genistein (4',5,7-trihydroxyisoflavone, G), genistin (genistein 7-O-β-D-glucoside, G7G), daidzein (4',7-dihydroxyisoflavone, D), daidzin (daidzein 7-O-β-Dglucoside, D7G), apigenin (4',5,7-trihydroxyflavone, A), and p-nitrophenyl β-D-glucopyranoside (pNPG) were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Apigenin 7-O-β-D-glucoside (A7G), quercetin (3,3',4',5,7-pentahydroxyflavone, Q), quercitrin (quercetin 3-O-α-L-rhamnoside, Q3R), and 1-deoxynojirimycin hydrochloride were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Triton® X-100 and D,L-dithiothreitol were purchased from Oriental Yeast Co., Ltd. (Osaka, Japan), Promega Co. (Madison, WI, USA), and Fluka (Buchs SG, Zurich, Switzerland), respectively. All other chemicals and solvents were of analytical grade. Extraction of flavonoids from gochujang Extraction 1: Samples (1.0 g) collected at intervals of 0, 6, 12, 18, 24, and 30 days during the gochujang manufacture were homogenized with 20 mL distilled water using a homogenizer (BM-2 Nissei bio-mixer; Nihonseiki Kaisha Ltd., Tokyo, Japan). The solution was partitioned with nhexane (20 mL, 3 times) and the n-hexane layer was discarded. The aqueous layer (20 mL) was successively partitioned with ethyl acetate (EtOAc, 20 mL) 3 times. The EtOAc layers were combined and concentrated in vacuo. The concentrate was dissolved in 1 mL of MeOH/EtOAc =1:1 (v/v) and filtered through a Millipore membrane (0.45 µm; Bedford, MA, USA). These samples prepared by extraction 1 were used for HPLC analysis of G7G, A7G, Q3R, G, A, Q, and D in the gochujang. Extraction 2: Samples (1.0 g) collected at intervals of 0, 6, 12, 18, 24, and 30 days during the gochujang manufacture were homogenized with MeOH (20 mL) using the same conditions described for extraction 1 and then filtered through no. 2 filter paper (Whatman International, Maidstone,

β-Glucosidase Inhibition Activity of Gochujang

UK). After filtration, the residue was extracted with 20 mL ethanol (EtOH) and filtered again. The MeOH and EtOH extracts were combined and concentrated in vacuo to remove organic solvents. The extract was suspended by adding 20 mL water. The water suspension was partitioned with n-hexane (20 mL, 3 times) and the n-hexane layer was discarded. The aqueous layer (20 mL) was successively partitioned with water-saturated n-butanol (BuOH, 20 mL, 3 times), and the BuOH layers were combined and concentrated in vacuo. The concentrate was dissolved with 1 mL of MeOH/EtOAc=1:1 (v/v) and filtered through a 0.45 µm Millipore membrane. Samples prepared by extraction 2 were used for HPLC analysis of D7G in gochujang. HPLC analysis of flavonoids in gochujang The samples (10 mg wet wt. eq.) prepared by extraction 1 and 2 of gochujang were analyzed by HPLC (Shimadzu, Kyoto, Japan), containing a photodiode array system (SPD-M20D; Shimadzu) operating at 200-800 nm. Flavonoids were separated on a Shim-pack Prep-ODS (H) KIT (4.6 mm i.d.×250 mm, 5 µm; Shimadzu) column using a linear gradient of 30% MeOH, containing 20 mM KH2PO4 (eluent A) to 58% MeOH (eluent B). The elution was increased to 25% B for 5 min, increased to 60% B at 25 min, increased to 63% D at 45 min, and held at 100% B until 55 min. Detection was accomplished at 254 nm, and the flow rate was 1.0 mL/min. Standard solutions (mg/mL) of flavonoids including G7G, D7G, A7G, Q3R, G, A, and Q in MeOH and D in EtOH were diluted to the appropriate concentrations to construct calibration curves. The calibration curves were constructed by plotting the peak areas vs. the concentration of each compound. Recovery of each compound was determined in triplicate using the spiking method. Flavonoid content in gochujang was qualified and quantified in triplicate experiments. Extraction of crude enzymes from gochujang Extraction of crude enzymes including β-glucosidase from gochujang was performed according to the method described by Iwata et al. (27), with slight modifications. Briefly, samples (20 g) were collected at intervals of 0, 6, 12, 18, 24, and 30 days during gochujang manufacture. The samples were homogenized with 100 mL of 10 mM acetate buffer (pH 5.0) containing 5 mM D,L-dithiothereitol and 90 mM NaCl. After standing at 4oC for 12 h, the homogenate was centrifuged at 8,000×g and 4oC for 15 min. The supernatant was mixed with ammonium sulfate (46 g) and centrifuged at 8,000×g and 4oC for 15 min. The precipitate was dissolved in 2 mL, 50 mM acetate buffer (pH 6.2). The crude enzyme solution was dialyzed for 3 h with a Pellicon membrane (12,000-14,000 Da; Millipore) with a molecular weight cut-off of 12,000 Da (200 mL, 3 times). Crude

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enzymes were recovered from the membrane with 50 mM acetate buffer (pH 6.2) and diluted to a total volume of 4 mL. The crude enzyme solution was used to measure βglucosidase activity. Determination of β-glucosidase activity in the gochujang crude enzyme extract The β-glucosidase activity of the crude enzyme solution prepared from gochujang was determined using pNPG as the substrate. Briefly, the crude enzyme solution (200 µL, gochujang 10 mg wet. wt. eq.) was mixed with 1 mL of 100 mM acetate buffer (pH 4.5) containing 3 mM pNPG. The mixture was incubated at 37oC for 100 min. The reaction was stopped by the adding EtOH (2 mL) and then centrifuged at 8,000×g for 15 min. The supernatant was filtered through a 0.45 µm membrane filter (Millipore). The p-nitrophenol (pNP) content liberated from the pNPG in the filtrate was analyzed by HPLC under the following conditions: column, ODS-80Ts (TSK-gel, 4.6 i.d.×250 mm, 5 µm; Tosoh, Tokyo, Japan); mobile phase, MeOH/H2O=35:75 (v/v); detection, 405 nm (SPD10A VP; Shimadzu); and flow rate, 1.0 mL/min. The pNP peak on the HPLC chromatogram was used as a measure of β-glucosidase activity. The maximum β-glucosidase activity of the gochujang samples was considered 100%, and the changes in β-glucosidase activity as a function of time during gochujang manufacturing were expressed as a relative percentage. This analysis was performed in triplicate. Determination of β-glucosidase inhibition activity of extracts of gochujang and its materials MeOH extracts of the final product of gochujang and its materials were respectively prepared from 1 g gochujang wet wt. equivalents [red pepper powder, 178 mg (17.8% in gochujang 1 g); wheat flour, 323 mg (32.3% in gochujang 1 g); meju, 18 mg (1.8% in gochujang 1 g); koji, 3 mg (0.3% in gochujang 1 g)]. The gochujang and its materials, which corresponded to 1 g wet wt. equivalents, were homogenized with MeOH (20 mL) and sonicated for 1 h. The slurries after the extraction were filtered (no. 2; Whatman) and the filtrates were concentrated in vacuo to give their MeOH extracts. Each MeOH extract was dissolved in 20 mL distilled water. The H2O suspension (20 mL) of each MeOH extract was successively partitioned with n-hexane (20 mL, 3 times), EtOAc (20 mL, 3 times), and BuOH (20 mL, 3 times), and each layer was concentrated in vacuo. n-Hexane and EtOAc layers were dissolved with 100 mM acetate buffer (pH 4.5) containing 5% Triton X100 and BuOH and H2O layers were dissolved with 100 mM acetate buffer (pH 4.5). Salt (101 mg, 10.1% in gochujang 1 g) was dissolved in 300 µL distilled water with the MeOH extract and partitioned to determine βglucosidase inhibition activity.

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The β-glucosidase inhibition activity in fractions obtained after solvent fractionation of gochujang and its material MeOH extracts were evaluated by reacting them with crude enzyme solutions obtained from gochujang. The crude enzyme solution, including β-glucosidase, was prepared at times when the gochujang samples displayed maximum activity during gochujang manufacturing. Each sample (100 µL, gochujang 0.5 g wet wt. eq.) extracted from gochujang materials was mixed with the crude enzyme solution (200 µL, 50 mM acetate buffer, pH 4.5) prepared from 1 g wet wt. gochujang. pNPG [final concentration, 3 mM; in 100 mM acetate buffer (pH 4.5)] as a substrate was also reacted with the crude enzyme solution under the same conditions as the gochujang samples. HPLC analysis to determine the librated pNP in the reaction solution was performed as described above. β-Glucosidase inhibition activity was directly proportional to the decrease in pNP peak area on the HPLC chromatogram of the reaction solution. That is, inhibition of β-glucosidase was measured based on the decrease in the pNP peak area on the HPLC chromatogram of the solution reacted with the crude enzyme and each extract of gochujang materials. A 0% inhibition was defined as the pNP peak area when the pNPG reaction solution (18.8 µM) and crude enzyme extracted from gochujang exhibited the highest βglucosidase activity during gochujang fermentation (1 g wet wt. eq.). 1-Deoxynojirimycin hydrochloride (final concentration, 50 µg), which is a β-glucosidase inhibitor, was used as a positive control (28). All analyses were performed in duplicate. Statistical analysis The data for individual flavonoid contents and β-glucosidase activity during gochujang manufacturing and β-glucosidase activities of gochujang ingredients were expressed as mean±standard deviation using the SPSS (version 17.0; IBM, Armonk, NY, USA). Statistical differences were analyzed by Duncan’s multiple comparison test (p0.999). In addition, G7G (93%), A7G (93%), Q3R (93%), G (96%), A (98%), Q (90%), and D (96%) in the EtOAc layer (Extraction 1) of the gochujang MeOH extract showed a high recovery of ≥90%. However, the recovery (15%) of D7G (tR 14.5 min) was lower than that of the other compounds. Therefore, the H2O suspension of combined gochujang MeOH and EtOH extracts was partitioned with BuOH after solvent fractionation with n-hexane to enhance extraction recovery of D7G. The BuOH layer was analyzed using the same HPLC conditions described above. When this extraction process was used, the recovery of D7G in the BuOH layer increased from 15% to 98% (Extraction 2 method) (Fig. 1C, down arrow). However, the other flavonoids in the BuOH layer (Extraction 2) showed lower recovery rates than those in the EtOAc layer (Extraction 1). Therefore, quantification of D7G was carried out using the Extraction 2 method and quantification of A7G, Q3R, G, A, Q, and D was accomplished using the Extraction 1 method. Changes in flavonoid contents during gochujang manufacturing Changes in the content of flavonoid

β-Glucosidase Inhibition Activity of Gochujang

Fig. 2. Change in the flavonoid content during gochujang manufacturing. ■, Daidzein; □, daidzin; ●, genistein; ○, genistin; △, quercitrin; ▽, apigenin 7-O-β-D-glucopyranoside. Data are expressed mean±SD of triplicate experiments.

glycosides and their aglycones with elapsed time during gochujang manufacturing was analyzed using the same HPLC conditions described above. The content of the four flavonoid glycosides (G7G, Q3R, A7G, and D7G) did not significantly change during gochujang manufacturing (Fig. 2), suggesting that flavonoid glycosides were not hydrolyzed during gochujang manufacturing. In addition, Q and A, the aglycones of Q3R and A7G, were not detected at any stage of gochujang manufacturing. It was clear that Q and A were not produced by hydrolysis of their glycosides (Q3R, A7G) during gochujang manufacturing. However, D (Fig. 2), the aglycone of D7G (Fig. 2), showed the highest content (75.3±3.6 mg/100 g gochujang wet wt. eq.) of flavonoids detected from gochujang. In addition, G (8.0±0.4 mg/100 g gochujang wet wt. eq.), the aglycone of G7G (Fig. 2), was also detected from gochujang, although its content was much lower in comparison with that of D. However, the D and G contents did not change significantly during gochujang manufacturing. Isoflavone aglycones (D and G) which are produced by hydrolysis of isoflavone glucosides (D7G and G7G), are abundantly present in fermented soybean products (29,30). Meju, a Korean fermented soybean paste (3), is used as one of the main ingredients in gochujang. Therefore, the aglycones, D and G, detected in gochujang were assumed to have originated from the meju. These results indicate that isoflavone glucosides including meju, were not hydrolyzed like Q3R and A7G contained in red pepper after mixing the various gochujang materials (7,15) during gochujang fermentation. Flavonoid glycosides contained in food materials are hydrolyzed to their aglycones during fermentation (4,29, 31,32). However, the flavonoid glycosides were not hydrolyzed to their aglycones during gochujang fermentation. There are two possible explanations for this result. First, β-

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Fig. 3. Change in relative β-glucosidase activity during gochujang manufacturing. The relative activity is expressed as the percentage of the maximum activity attained from the βglucosidase assay. Data are expressed mean±SD of triplicate experiments. a-dDifferent letters indicated a significant difference (pkoji (10.3%)>meju (5.0%)>wheat (0%) (Fig. 6). Thus, salt and red pepper more strongly inhibited β-glucosidase activity than that of the other materials. High salt content (about 10%) can inhibit β-glucosidase activity generated by A. oryzae during the fermentation of douchi, which is a fermented soybean curd used in China (33). In addition, those authors reported that the content of hydrolyzed aglycones (G and D) is inversely proportional to the NaCl content (33). In the present study, the salt content (10.1%) was similar to previously reported salt concentrations (about 10%). Nevertheless, in this study, isoflavone glucosides (G7G and D7G) and other flavonoid glycosides (A7G and Q3R) were not hydrolyzed during gochujang manufacture. This indicates that other β-glucosidase inhibitor(s) in addition to salt is (are) contained in gochujang as inhibition of β-glucosidase activity was also observed for the extracts of red pepper, koji, and meju. In particular, among them, the activity of the H2O layer of the red pepper extract was comparable to that of the salt solution. Therefore, we further evaluated the β-glucosidase inhibition activity of layers obtained after solvent-fractionation of the red pepper MeOH extract (gochujang 1 g wet wt. eq.) to better understand the characteristics of the β-glucosidase inhibitor contained in the red pepper extract. The BuOH and H2O layers showed very high inhibition activities of 66.2% and 90.1% (data not shown), respectively, which was similar to

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the layers obtained after solvent-fractionation of the gochujang MeOH extract (Fig. 5). Thus, the red pepper contained (a) hydrophilic inhibitor(s) of β-glucosidase. The presence of a β-glucosidase inhibitor in pepper and gochujang has never been reported. Therefore, isolation and structural determination of β-glucosidase inhibitor(s) contained in pepper is currently underway. Glycosidases present in plant materials affect aroma (34,35), taste (36), and functionality (37) of foods (38,39). Therefore, the presence and absence of secretion and activity of glycosidic hydrolase by microorganisms in relation to food fermentation may be a very important factor to control aroma, taste, and functionality of fermented foods. The results of the present study are expected to provide useful information to improve the quality and biological activity of gochujang.

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