Chiang Mai J. Sci. 2016; 43(1) : 158-168 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Efficient Synthesis of 4-Vinyl Guaiacol via Bioconversion of Ferulic Acid by Volvariella volvacea Keerati Tanruean [a,b] and Nuansri Rakariyatham*[b,c] [a] Division of Biotechnology, Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. [c] Faculty of Health Science, Nation University, Lampang 52000, Thailand. *Author for correspondence; e-mail: [email protected]

Received: 8 March 2014 Accepted: 7 July 2014

ABSTRACT Ferulic acid is a phenolic compound that is extremely abundant in the cell walls of plants. It has been of interest for use as a starting material in the bioconversion process of subsequent highly valuable compounds. In this study, Volvariella volvacea was investigated for its ability to convert ferulic acid to various products of degradation (4-vinyl guaiacol, vanillic acid and vanillyl alcohol) by HPLC and LC-DAD-ESIMS. The results showed that the mycelium of V. volvacea had potential to produce high amounts of 4-vinyl guaiacol (88.2 mg/L), vanillic acid (59.1 mg/L) and vanillyl alcohol (39.7 mg/L) at 42, 42 and 96 hours of growth on ferulic acid medium, respectively. Moreover, the effects of various sulfhydryl compounds and their derivatives of sulfur containing amino acids: cysteine, cysteine hydrochloride monohydrate, dithiothreitol, glutathione and methionine treatment on ferulic acid bioconversion were also investigated. Most all of the sulfhydryl compounds, except dithiothreitol, could enhance 4-vinyl guaiacol production; especially cysteine hydrochloride monohydrate which displayed a 47.9% increase in 4-vinyl guaiacol production (136.7 mg/L) when compared with the control. This is the first report of the bioconversion of ferulic acid into 4-vinyl guaiacol V.by volvacea.

Key words: 4-vinyl guaiacol, ferulic acid, edible mushroom

1. INTRODUCTION Ferulic acid (4-hydroxy-3-methoxycinnamic the most important aromatic flavor compound acid; Figure 1A) is an abundant type of used in foods, beverages, pharmaceuticals hydroxycinnamic acid that is found in the and perfumes [2]. Due to the high price and cell wall of plants, either in its free form or low availability of natural vanillin and other covalently linked to the biopolymers. It is flavor aromatic compounds, and the trend well known for its antioxidant properties and toward natural flavors, an extensive research has been widely used in the food industry [1]. study on the production of natural vanillin Moreover, it is also used as a starting material and other flavor aromatic compounds by the in its bioconversion to vanillin (4-hydroxy-3- biotechnological process has been initiated methoxybenzaldehyde; Figure 1B), which is [3,4]. Various microorganisms have been Chiang Mai J. Sci. 2016; 43(1) 159

used to degrade ferulic acid into vanillin and may affect one or more enzymatic steps in other flavor aromatic compounds via different the bioconversion pathway depending on the metabolic routes [5]. nature of the microorganisms [5,11]. Apart from vanillin, ferulic acid has also Among different types of microorganisms been used to generate other highly valuable used in the bioconversion of phenolic monomers compounds, such as 4-vinyl guaiacol (3-methoxy- into high value aromatic compounds, white rot 4-hydroxystyrene; Figure 1C), vanillic acid fungi seem to be advantageous [12,13]. Gupta (4-hydroxy-3-methoxy benzoic acid; Figure et al. [14] reported that Sporotrichum pulverulentum 1D) and vanillyl alcohol (4-hydroxy-3-methoxy- could convert ferulic acid to coniferyl aldehyde, benzyl alcohol; Figure 1E). 4-Vinyl guaiacol is dihydroferulic acid, dihydroconiferyl alcohol, a volatile phenol with a spicy clove-like aroma, vanillic acid and methoxy hydroquinone. which can be obtained from the decarboxylation Moreover, Tsujiyama and Ueno [15] suggested of ferulic acid molecules. It has a 40-times that an edible mushroom, Schizophyllum commune, higher commercial price than ferulic acid [6], could convert ferulic acid into 4-vinyl guaiacol and is used for flavoring in beers, wine and soy and this could then be oxidized into sauce [7,8]. Similarly, vanillic acid and vanillyl vanillin and vanillic acid. However, there have alcohol, an oxidized and reduced form of been a limited number of reports on the role vanillin are the main intermediates in lignin and of edible mushrooms in the production of ferulic acid degradation, and they are usually ferulic acid metabolites, especially Volvariella used as flavoring compounds [9,10]. Regarding volvacea. Therefore, it is of great interest to metabolite production, Labuda et al. [11] has use ferulic acid as a of the edible attempted to enhance the production yield of mushroom, V. volvacea, in the production of vanillin using sulfhydryl compounds during the high-value added ferulic acid metabolites. In bioconversion of ferulic acid by Pseudomonas addition, the use of sulfhydryl compounds to putida ATCC 55180. However, the mechanism enhance bioconversion products from ferulic of sulfhydryl compounds on the bioconversion acid was also investigated. of ferulic acid has not been well understood. These compounds can act as antioxidants or 2. MATERIALS AND METHODS reducing reagents via several mechanisms, such 2.1 Chemicals as metal chelaters or radical quenchers, which Ferulic acid (trans-, 99%), 4-vinylguaiacol

(A) (B)

(C) (D) (E) Figure 1. Structure of (A) ferulic acid, (B) vanillin, (C) 4-vinyl guaiacol, (D) vanillic acid and (E) vanillyl alcohol. 160 Chiang Mai J. Sci. 2016; 43(1)

(≥98%), vanillyl alcohol (98%), vanillin (99%), collected every 6 hours for first 48 hour, then glutathione reduced (≥98%) were purchased every 12 hours over a 120-hour period. The from Sigma-Aldrich. Vanillic acid (≥97%) and fermented broth was filtered using Whatman L-cysteine hydrochloride monohydrate (≥99%) no. 1, and was then diluted with an equal were obtained from Fluka. DL-1,4-dithiothreitol volume of methanol (HPLC grade). After that, (99%) and L(+) cysteine (99%) were purchased the samples were filtered quickly through a 0.2 from Acros Organics. DL-methionine (99.5%) µm membrane filter, and applied to HPLC and was obtained from Himedia and potato dextrose LC-DAD-ESIMS. agar (PDA) was obtained from Difco. All the The percent conversion of ferulic acid solvents used for high performance liquid was expressed as follows: chromatography (HPLC) were of the HPLC (FA0 FAf) grade and all other chemicals were of the % conversion = × 100 FA0 analytical grade. − Where FA0 and FAf are the initial and final 2.2 Mushroom Maintenance concentrations of ferulic acid (mg/L), respectively. The mycelium of Volvariella volvacea no. 6, was purchased from the Biotechnology 2.4 Analytical Methods Research and Development Office, Department 2.4.1 High performance liquid chromatography of Agriculture, Ministry of Agriculture and analysis Cooperative, Bangkok, Thailand. The fungal The HPLC separation of ferulic acid, strain was grown and maintained on PDA 4-vinyl guaiacol, vanillic acid, vanillyl alcohol slants and incubated at ambient temperature. and vanillin was performed using the protocol described previously by Xie et al. [17] with 2.3 Bioconversion of Ferulic Acid in Basal slight modifications on an Agilent 1100 series Medium equipped with a binary pump. The used column The analysis of the bioconversion products was a Zorbax SB-C18, 5 µm (4.6x150 mm) (4-vinyl guaiacol, vanillic acid, vanillyl alcohol type from Agilent USA. The fingerprints were and vanillin) from ferulic acid by V. volvacea in recorded at an optimized wave length of 280 the basal medium will be analyzed. The basal nm. A linear gradient of two solvents was used: medium for growth of the fungal strain was solvent A (0.5% acetic acid in water, v/v), and prepared as described previously [16], which solvent B (acetonitrile). The linear gradient was contained maltose 20 g/L, ammonium tartrate run at 25°C in 15 min from 5% to 20% and in

1.8 g/L, yeast extract 0.5 g/L, MgSO4∙7H2O 15−40 min from 20−40% of B, at a flow rate

0.5 g/L, K2HPO4 0.2 g/L, CaCl2 1.3 mg/L and of 0.8 mL/min. The injection volumes for all

VB2 2.5 mg/L. The fungal strain was grown samples were 20 µL. The solvent solutions on potato dextrose agar plates at ambient were vacuum-degassed with ultrasonication temperature for 7 days and three discs (6 mm prior to usage. The samples and standards were diameter) of mycelia containing agar were filtered quickly through a 0.2 µm membrane inoculated in 125 mL flasks containing 25 mL filter. The degradation products were identified basal medium, and incubated at 30°C and 200 by comparison with the retention times of all rpm. After 48 hours of incubation, ferulic acid standard compounds. Quantification of the (1 mM final concentration) was added in to the degradation products were calculated by using medium. The bioconversion was carried out at calibration curves prepared from the HPLC 30°C and 200 rpm. 0.5 mL of the sample was peak areas of each standard. Chiang Mai J. Sci. 2016; 43(1) 161

2.4.2 LC-DAD-ESIMS analysis concentration of sulfhydryl compound used that LC-DAD-ESIMS was conducted on an was effective in the bioconversion process [11]) Agilent Hewlett Packard 1100 series equipped were individually added in to the medium. The with a binary pump and an auto sampler. The bioconversion was then carried out at 30°C and LC separation was achieved using inertsil ODS-3 200 rpm. The samples were collected every 24 (5 µm, 2.1x150 mm) at a flow rate of 0.3 mL/ hours over a 120-hour period. The fermented min. The chromatograms were recorded at 280 broth was filtered using Whatman no. 1, and and 330 nm using a linear gradient of the two extracted with an equal volume of methanol solvents: solvent A (0.1% formic acid in water, (HPLC grade). The samples were then filtered v/v), and solvent B (methanol). The linear quickly through a 0.2 µm membrane filter, and gradient was run at 25°C from 25% to 45% applied to HPLC. over 10 min, from 45−65% over 10−20 min and held at 65% until 30 min, and then reduced 2.6 Statistical Analysis from 65% to 25% at 32 min of solvent B. The The results of all experiments were expressed analyses were done in a positive ion mode base as mean ± standard deviation. Analysis of on the results from the standard compounds. variance was performed by ANOVA procedures The conditions for mass spectrometry were and Scheffe’s multiple comparison test was used as follows: drying gas (nitrogen) flow 8.0 L/ for significant differences P<0.05( ) identified min; nebulizer flow 35; drying gas temperature between treatments. 320°C; capillary voltage 4000 V. The masses were scanned at a range of 50 to 300 with 3. RESULTS AND DISCUSSION a fragmentor of 80. The injection volumes The fungus, Volvariella volvacea, could degrade of all samples were 10 µL. The samples and ferulic acid and a 100 percent conversion of 1 standards (4-vinyl guaiacol, vanillic acid and mM ferulic acid was found at 48 hours (Figure vanillyl alcohol) were filtered quickly through 2 and Figure 3). The products derived from V. a 0.2 µm membrane filter and subjected to LC- volvacea extract by HPLC revealed a significant DAD-ESIMS analysis. The chromatograms and amount of ferulic acid degradation products fragmentation patterns served as an in-house (4-vinyl guaiacol, vanillic acid and vanillyl alcohol) reference resource. (Figure 3). Moreover, the formation of these products that were found in the medium of 2.5 Effects of Sulfhydryl Compounds on V. volvacea were confirmed using an in-house Bioconversion of Ferulic Acid library source of reference for the fragmentation To determine the effects of sulfhydryl pattern of each compound and they were then compound and their derivatives of sulfur compared with the authentic standard by LC- containing amino acids (cysteine, cysteine DAD-ESIMS (Figure 4). A summary of the hydrochloride monohydrate, dithiothreitol, LC-MS data of vanillyl alcohol, vanillic acid and glutathione and methionine), three discs (6 mm 4-vinyl guaiacol standards, and the characterized diameter) of mycelia containing agar of 7 days results of the ferulic acid degradation products culture of V. volvacea were separately inoculated in the V. volvacea medium are shown in Table 1. in 250 mL flask containing 25 mL basal medium. Based on the results in Table 1, it is suggested The media were incubated at 30°C and 200 that the compounds eluting at the retention rpm. After 48 hours of incubation, ferulic times of 4.69 (compound 1), 9.26 (compound acid (1 mM final concentration) and 5 mM 2) and 20.2 (compound 3) min correspond of each sulfhydryl compound (the minimum to standard vanillyl alcohol, vanillic acid and 162 Chiang Mai J. Sci. 2016; 43(1)

Ferulic acid Vanillic acid 4-Vinyl guaiacol Vanillyl alcohol 200 100

180

160 80

140

120 60

100

80 40

60 Ferulic acid concentration (mg/L) concentration acid Ferulic Products concentration (mg/L) 40 20

20

0 0 0 6 12 18 24 30 36 42 48 60 72 84 96 108 120

Time (hour)

Figure 2. Ferulic acid degradation and the production of 4-vinyl guaiacol, vanillic acid and vanillyl alcohol by V. volvacea.

mAU A. mAU B. 350 350 Ferulic acid 300 300

250 250 Ferulic acid

200 200

150 150

100 100 Vanillic acid 4-Vinyl guaiacol 50 Vanillic acid 4-Vinyl guaiacol 50

0 0 0 5 10 15 20 25 30 35 min 0 5 10 15 20 25 30 35 min

mAU mAU C. D. 350 350

300 300

250 250 Ferulic acid 200 200 Ferulic acid 150 150 Vanillic acid 4-Vinyl guaiacol 100 Vanillic acid 4-Vinyl guaiacol 100

50 50

0 0 0 5 10 15 20 25 30 35 min 0 5 10 15 20 25 30 35 min

mAU mAU 350 E. 350 F. 300 300

250 250

200 200

150 Vanillic acid 4-Vinyl guaiacol 150 Vanillic acid 4-Vinyl guaiacol

100 100 Vanillyl alcohol Ferulic acid Vanillyl alcohol 50 50

0 0 0 5 10 15 20 25 30 35 min 0 5 10 15 20 25 30 35 min

Figure 3. Overlay of HPLC chromatograms at 280 nm of V. volvacea medium at (A) 18 hours (B) 24 hours (C) 30 hours (D) 36 hours (E) 42 hours and (F) 48 hours. HPLC: column Zorbax SB-C18, 5 µm (4.6x150 mm), gradient solution (A) 0.5 % acetic acid and (B) acetronitrile 100 % (B. 5 - 20 %, 15 min and 20 - 40 %, 15-40 min), UV detector 280 nm, flow rate 0.8 mL/min and injection volume 20 µL. Chiang Mai J. Sci. 2016; 43(1) 163 1 . 169 1 . mAU mAU A. B. 100 100 137 1 80 .

500 125

80 1

500 .

60 93 60 2 400 400 40 .

40 151 20 1 1 . .

20 1 300 1

300 . 138 60 122 94 . 0 m/z 0 m/z 50 100 150 200 250 200 50 100 150 200 250 200

100 100

0 0 0 5 10 15 20 25 30 35 min 0 5 10 15 20 25 30 35 min 1 .

100 151 mAU C. D. 80 mAU

500 60 250 1 40 . 1 . 400 119 91 20 200

300 0 m/z 150 50 100 150 200 250 200 100

100 50

0 0 0 5 10 15 20 25 30 35 min 0 5 10 15 20 25 30 35 min

Figure 4. Overlay of LC chromatogram, mass spectrum and structure of the authentic standard of (A.) vanillyl alcohol, (B.) vanillic acid, (C.) 4-vinyl guaiacol and (D) V. volvacea medium. LC: column inertsil ODS-3 (5 µm, 2.1x150 mm), gradient solution (A) 0.1 % formic acid and (B) methanol 100 % (B. 25 - 45 %, 10 min; 45 - 65 %, 10-20 min; 65%, 20-30 min and 65 - 25%, 30-32 min), UV detector 280 nm, flow rate 0.3 mL/min and injection volume 10 µL. MS: drying gas (nitrogen), flow rate 8.0 L/min, temperature 320°C, nebulizer flow 35, capillary voltage 4000 V, mass scan 50-300, fragmentor 80.

4-vinyl guaiacol, respectively. The compound The potential of V. volvacea in the eluting at 4.69 min had an abundant ion level bioconversion of ferulic acid to the highest of measurement at m/z 137 (100) and another concentration of 4-vinyl guaiacol, vanillic acid ion measurement at m/z 138 (12), 122 (8) and and vanillyl alcohol products is shown in Table 60 (7), corresponding to the mass spectral 2. The reduction of ferulic acid concentration pattern of vanillyl alcohol, which contained an (46.6%) was noticed in the first 24 hours and it abundant ion measurement at m/z 137 (100) and was degraded completely (100%) at 48 hours a secondary ion measurement at m/z 138 (10), after ferulic acid supplementation (Figure 2). 122 (7), 94 (7), 60 (7). On the other hand, the The production of 4-vinyl guaiacol and vanillic compound eluting at 9.26 min had an abundant acid increased steadily over the first twelve hours ion measurement at m/z 169 (100), 93 (72), (24.0 mg/L (0.16 mM) and 6.40 mg/L (0.04 mM), 125 (70), 183 (37), 151 (32), corresponding to respectively) after ferulic acid supplementation the mass spectral pattern of vanillic acid (m/z and this reached maximum concentrations of 169 (100), 125 (67), 93 (58), 151 (29)). While, 88.2 mg/L (0.59 mM) and 59.1 mg/L (0.35 the compound eluting at 20.2 min showed the mM) at 42 hours of incubation, respectively. major ion measurement at m/z 151 (100), 119 Additionally, vanillyl alcohol started to accumulate (30), 91 (24), which was the same as the mass at the 42-hour mark (15.81 mg/L; 0.10 mM) spectral pattern of 4-vinyl guaiacol (151 (100), of incubation and reached the maximum 119 (32), 91 (26)). concentration of 39.7 mg/L (0.26 mM) at 96 164 Chiang Mai J. Sci. 2016; 43(1)

Table 1. Summary of LC-MS results of vanillyl alcohol, vanillic acid and 4-vinyl guaiacol standards and the components in the V. volvacea medium (compound 1, 2 and 3).

Molecular MS retention time Compounds M/Z (abundance) weight (min) Vanillyl alcohol 154 4.66 137 (100), 138 (10), 122 (7), 94 (7), 60 (7) Vanillic acid 168 9.30 169 (100), 125 (67), 93 (58), 151 (29) 4-Vinyl guaiacol 150 20.1 151 (100), 119 (32), 91 (26) 1 - 4.69 137 (100), 138 (12), 122 (8), 60 (7) 2 - 9.26 169 (100), 125 (70), 93 (72), 183 (37),151 (32) 3 - 20.2 151 (100), 119 (30), 91 (24)

Table 2. Products of ferulic acid conversion by V. volvacea.

Product Products concentration (mg/L)* Ferulic acid conversion (%) Time (hour)

4-Vinyl guaiacol 88.2±1.5a 92.5 42

Vanillic acid 59.1±0.9b 92.5 42

Vanillyl alcohol 39.7±0.8c 100 96 *Average ± standard deviation from three replicates. The different letters in the same column are significantly different according to Scheffe’s multiple comparison test (P<0.05). hours of incubation. Moreover, the production be concluded that in our study on V. volvacea, of vanillyl alcohol increased steadily, and was ferulic acid could be converted to 4-vinyl coupled to the decline of 4-vinyl guaiacol and guaiacol and vanillic acid. While, vanillyl alcohol vanillic acid. Based on these results, it was might has been formed via vanillic acid upon noticed that ferulic acid could be degraded the consumption of ferulic acid. In a previous by V. volvacea and could then generate 4-vinyl study, Tsujiyama and Ueno [15] suggested that guaiacol as the major degradation products, Schizophyllum commune could convert ferulic acid which showed statistically higher contents into 4-vinyl guaiacol by decarboxylation prior than vanillic acid and vanillyl alcohol (P<0.05). to the formation of vanillin and vanillic acid, Donaghy et al. [18] reported that Candida lambica respectively. Additionally, Overhage et al. [21] could convert ferulic acid into 4-vinyl guaiacol revealed that vanillin was converted to vanillic using the ferulic acid decarboxylase. acid by vanillin dehydrogenase in Pseudomonas sp. Although, vanillic acid and vanillyl alcohol strain HR199. In contrast, Falconnier et al. [22] (an oxidized and reduced form of vanillin) demonstrated that in white rot fungus, Pycnoporus were also found as degradation products by cinnabarinus I-937, the propeonic side chain of V. volvacea, no accumulation of vanillin in the ferulic acid molecules was oxidatively cleaved to culture medium was observed over 120 hours. generate vanillic acid during the bioconversion This may be due to the ability of the vanillin process, and then could further be reduced to intermediate either to be quickly oxidized or to vanillin and vanillyl alcohol, respectively. By this convert to other compounds [19,20]. It could bioconversion, the two reduction steps might Chiang Mai J. Sci. 2016; 43(1) 165

involve aryl and aryl mM). These results suggest that vanillyl alcohol alcohol dehydrogenase, respectively. could be oxidized to vanillin, which might In order to study the possible pathway of then be converted to vanillic acid. Thus, it was the ferulic acid metabolism of V. volvacea, 1 mM assumed that V. volvacea might produce the of each product: 4-vinyl guaiacol, vanillic acid relevant enzyme involved with the oxidation of or vanillyl alcohol, were individually supplied to vanillyl alcohol and vanillin. When vanillin was the medium as the sole carbon source, instead supplied to the medium as a sole carbon source, of ferulic acid. The degradation products were vanillic acid and vanillyl alcohol were found at detected by HPLC every 24 hours over a 120- the concentrations of 43.7% (51.8 mg/L; 0.31 hour period. When 4-vinyl guaiacol was used mM) and 40.2% (46.8 mg/L; 0.30 mM) after as the sole carbon source, vanillic acid, vanillyl 120 hours of incubation, respectively, while alcohol or other products were not detected 16.7% (23.8 mg/L; 0.16 mM) vanillin remained in the medium, while 40% (52.9 mg/L; 0.35 in the medium. These results suggested that mM) of 4-vinyl guaiacol remained after 120 V. volvacea might produce the relevant hours. These results indicated that 60% (79.0 involved with the oxidation of vanillin into mg/L; 0.53 mM) of 4-vinyl guaiacol may be vanillic acid, and also produced the catalytic used as the sole carbon source for growth, and enzyme that metabolized the conversion of no relevant enzyme can catalyze the conversion vanillin to vanillyl alcohol. Based on our results, of 4-vinyl guaiacol to vanillic acid, vanillyl the proposed metabolic pathway of ferulic alcohol or vanillin. When vanillic acid was used acid by V. volvacea has been shown in Figure 5. as the sole carbon source instead of ferulic Table 3 shows various effects of acid, 64.4% (95.3 mg/L; 0.62 mM) of vanillyl sulfhydryl compounds and their derivatives alcohol was formed as the degradation product, of sulfur containing amino acids, including while 33.4% (49.4 mg/L; 0.29 mM) of vanillic cysteine, cysteine hydrochloride monohydrate, acid remained after 120 hours of incubation. dithiothreitol, glutathione and methionine, on These results revealed that V. volvacea might the production of 4-vinyl guaiacol from the produce the catalytic enzyme involved with the ferulic acid bioconversion by V. volvacea. Most of reduction of vanillic acid into vanillyl alcohol, the sulfhydryl compounds supplemented in the but might not generate the relevant enzyme medium showed statistically higher production that catalyzed the conversion of vanillic acid to levels of 4-vinyl guaiacol concentrations than vanillin. When vanillyl alcohol was used as the the control (P<0.05), while the other products sole carbon source, it was rapidly metabolized to (vanillic acid and vanillyl alcohol) showed vanillin and reached a maximum concentration lower concentration levels than the control. of 62.4% (105.4 mg/L; 0.69 mM) at 48 hours, Supplementation of cysteine hydrochloride while 18.8% (31.7 mg/L; 0.19 mM) of vanillic monohydrate in the medium could increase acid was also found and 15.8% (26.6 mg/L; 4-vinyl guaiacol by 47.9% (136.7 mg/L; 0.17 mM) of vanillyl alcohol remained in the 0.91 mM) when compared with the control. medium. After that, vanillin was reduced to The supplementation of other substances, 11.60% (15.3 mg/L; 0.10 mM) at 120 hours glutathione, methionine and cysteine in the of incubation, while vanillic acid was found medium, exhibited higher concentrations of to increase steadily and exhibited 64.4% (84.7 4-vinyl guaiacol of 119.2 mg/L (0.79 mM), mg/L; 0.50 mM). Moreover, the original sole 103.5 mg/L (0.69 mM) and 101.1 mg/L (0.67 carbon source of vanillyl alcohol was also mM), respectively. Whereas, dithiothreitol could found to remain at 21.6% (36.2 mg/L; 0.23 degrade ferulic acid at only 29.2% and showed 166 Chiang Mai J. Sci. 2016; 43(1)

Ferulic acid 4-Vinyl guaiacol

Vanillyl alcohol

Vanillic acid

Vanillin

Figure 5. Proposed pathway of ferulic acid metabolism by V. volvacea.

Table 3. Effects of sulfhydryl compounds on the products from ferulic acid conversion by V. volvacea.

Products concentration (mg/L)* Treatment (sulfhydryl compounds) 4-Vinyl guaiacol Vanillic acid Vanillyl alcohol

Control 92.4±1.3d 54.9±1.4a 47.0±1.8a

Cysteine 101.1±1.3c 19.8±0.8b 18.3±0.1c

Cysteine hydrochloride a d c,d 136.7±2.4 5.9±0.4 16.5±0.1 monohydrate

Dithiothreitol ND ND ND

Glutathione 119.2±0.7b 9.7±0.1c 15.3±0.1d

Methionine 103.5±2.9c 9.8±0.1c 22.2±0.1b *Average ± standard deviation from three replicates. The different letters in the same column are significantly different according to Scheffe’s multiple comparison test (P<0.05). ND = not detected. Chiang Mai J. Sci. 2016; 43(1) 167

no accumulation of 4-vinyl guaiacol, vanillic ACKNOWLEDGEMENTS acid and vanillyl alcohol after 120 hours. The We are grateful to Center of Excellence effects of these sulfhydryl compounds on for Innovation in Chemistry (PERCH-CIC), 4-vinyl guaiacol production may come from Commission on Higher Education, Ministry of the inhibition effect of enzymes by directly Education and Department of Chemistry, Faculty bonding with copper at the of the of Science and Graduate School, Chiang Mai enzyme [23,24]. This may have affected the University, Thailand for the financial support. oxidation step of ferulic acid bioconversion, which then resulted in the reduction of vanillic REFERENCES acid and vanillyl alcohol. Moreover, sulfhydryl [1] Ou S. and Kwok K.C., J. Sci. Food Agric., compound may play a role as a catalyst for 2004; 84: 1261-1269. DOI 10.1002/ the rate of ferulic acid decarboxylation [25], jsfa.1873. which may result in the accumulation of 4-vinyl guaiacol. [2] Priefert H., Rabenhorst J. and Steinbuchel Based on our results, V. volvacea is considered A., Appl. Microbiol. Biotechnol., 2001; 56: an edible mushroom with a high potential in 296-314. DOI 10.1007/s002530100687. the production of 4-vinyl guaiacol (0.59 mM) [3] Nazareth S. and Mavinkurve S., Can. J. from ferulic acid (1 mM) when compared Microbiol., 1986; 32: 494-497. DOI 10.1139/ with the other edible mushrooms, such as S. m86-090. commune, which could produce only about 0.047 mM of 4-vinyl guaiacol [15]. Moreover, the [4] Huang Z., Dostal L. and Rosazza J.P., J. production of 4-vinyl guaiacol from ferulic Bacteriol., 1994; 176: 5912-5918. acid by V. volvacea increased to 0.91 mM when [5] Mathew S. and Abraham T.E., Crit. supplemented with cysteine hydrochloride Rev. Microbiol., 2006; 32: 115-125. DOI monohydrate. 10.1080/10408410600709628. [6] Landete J.M., Rodriguez H., Curiel J.A., de 4. CONCLUSIONS las Rivas B., Mancheno J.M. and Munoz This study demonstrated the potential of R., J. Ind. Microbiol. Biotechnol., 2010; 37: V. volvacea in the conversion of ferulic acid, the 617-624. DOI 10.1007/s10295-010-0709-6. main phenolic compound in plant cell walls, into high value metabolites. V. volvacea exhibited a [7] Mathew S., Abraham T.E. and Sudheesh high potential in ferulic acid bioconversion to S., J. Molecular Catal. B: Enzym., 2007; 44: 50.6% of 4-vinyl guaiacol (0.59 mM), 33.9% 48-52. DOI 10.1016/jmolcatb.2006.09.001. of vanillic acid (0.35 mM) at 42 hours of [8] Max B., Carballo J., Cortes S. and Dominguez incubation and 20.9% of vanillyl alcohol (0.26 J.M., Appl. Biochem. Biotechnol., 2012; 166: mM) at 96 hours of incubation. Moreover, 289-299. DOI 10.1007/s12010-011-9424-7. sulfhydryl compounds and their derivatives have been shown to have the potential to [9] Kim S.J., Kim M.C., Um J.Y. and Hong increase 4-vinyl guaiacol production from S.-H., Molecules, 2010; 15: 7208-7217. DOI ferulic acid, in which cysteine hydrochloride 10.3390/molecules15107208. monohydrate could enhance the production of [10] Hsu L.C., Wen Z.H. and Lee K.Y., US Pat. 4-vinyl guaiacol to 136.7 mg/L (0.91 mM) at 72 No. 0258951A1 (2009). hours of incubation, which corresponded to a [11] Labuda I. M., Goers S. K. and Keon K.A., 47.9% increase as compared with the control. US Pat. No. 5128253 (1992). 168 Chiang Mai J. Sci. 2016; 43(1)

[12] Kirk T.K., Annu. Rev. Phytopathol., 1971; [20] Fitzgerald D.J., Stratford M. and Narbad 9: 185-210. DOI 10.1146/annurev. A., Int. J. Food Microbiol., 2003; 86: 113-122. py.09.090171.001153. DOI 10.1016/s0168-1605(03)00059-x. [13] Abraham B.G. and Berger R.G., J. Agric. [21] Overhage J., Priefert H., Rabenhorst Food Chem., 1994; 42: 2344-2348. DOI J. and Steinbuchel A., Appl. Microbiol. 10.1021/jf00046a050. Biotechnol., 1999; 52: 820-828. DOI 10.1007/ s002530051598. [14] Gupta J., Hamp S., Buswell J. and Eriksson K.-E., Arch. Microbiol., 1981; 128: 349-354. [22] Falconnier B., Lapierre C., Lesagemeessen DOI 10.1007/bf00405911. L., Yonnet G., Brunerie P., Colonnaceccaldi B., Corrieu G. and Asther M., J. Biotechnol., [15] Tsujiyama S. and Ueno M., Biosci. Biotechnol. 1994; 37: 123-132. DOI 10.1016/0168- Biochem., 2008; 72: 212-215. DOI 10.1271/ 1656(94)90003-5. bbb.60606. [23] Valero E., Varon R. and Garcia-Carmona [16] Zheng L., Zheng P., Sun Z., Bai Y., Wang J. F., Biochem. J., 1991; 277: 869-874. and Guo X., Bioresour.Technol., 2007; 98: 1115- 1119. DOI 10.1016/j.biotech.2006.03.028. [24] Park Y.D., Lee S.J., Park K.H., Kim S.Y., Hahn M.J. and Yang J.M., J. [17] Xie C.Y., Gu Z.X., You X., Liu G., Tan Y. Protein Chem., 2003; : 613-623. DOI and Zhang H., Enz. Microb. Technol., 22 10.1023/b:jopc.0000008726.99095.48. 2010; 46: 125-128. DOI 10.1016/j. enzmictec.2009.10.005. [25] Huang H.K., Chen L.F., Tokashiki M., Ozawa T., Taira T. and Ito S., AMB Express, [18] Donaghy J.A., Kelly P.F. and McKay A., J. Sci. 2012; 2: 1-10. DOI 10.1186/2191-0855-2-4. Food Agric., 1999; 79: 453-456. DOI 10.1002/ (sici)1097-0010(19990301)79:3<453::aid- jsfa284>3.0.co;2-h. [19] Stentelaire C., Lesage-Meessen L., Oddou J., Bernard O., Bastin G., Ceccaldi B.C. and Asther M., J. Biosci. Bioeng., 2000; 89: 223- 230. DOI 10.1016/s13891723(00)88823-4.