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 product 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 substrate 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.