Natural Vanillin Production from Isoeugenol by Using Pseudomonas putida in Biphasic Bioconversion Medium
Abstract
Vanillin, is one of the most demanded flavoring agents in the world. Because of insufficient supply of natural vanillin, market demand is usually supplied by synthetic ones. In this study, it was investigated possibility of usage biphasic system in bioconversion of isoeugenol to vanillin by Pseudomonas putida (HUT 8100). Organic phase was composed of isoeugenol while biocatalyst, P. putida culture, was dispersed in aqueous phosphate solution. Isoeugenol was used as sole carbon source in concentrations ranging between 50–600 g L-1. Incubation was performed at 28 ○C, at pH 6.3 and 180 rpm shaking. Effect of initial substrate concentration and bioconversion time were investigated. Isoeugenol and vanillin amounts in medium were simultaneously analyzed in HPLC system. After 120 h incubation, vanillin reached the its highest level when 400 g L-1 isoeugenol was applied in medium. In specified conditions, it was achieved to produce 11.95 g L-1 vanillin with 6.2% molar yield within 15 days of bioconversion. It is thought that, obtained result by using biphasic system is very important for the industrial applications in production of natural vanillin via bioconversion.
- Introduction
Vanillin (4-hydroxy-3-methoxybenzaldehyde) which naturally occurs in vanilla orchid pods, is one of the most important flavoring compounds (Singh et al. 2015). It is widely used in food, beverages, perfume and pharmaceutical industries (Zhao et al. 2018). However, vanillin derived from the pods serves less than 1% of global market volume (Singh et al. 2015). 85% of annual vanillin production is came from a petro-based compound, guaiacol, and the rest is synthesized from lignin (Zhu et al. 2018). Market value of synthetic vanillin is only about 10–20 USD kg-1 while vanillin derived from Vanilla spp. is 1500 USD kg-1 (van Leeuwen et al. 2018). Although there is a distinct price advantage of the synthetic counterparts, consumer interests to natural compounds, that considered as healthy, has forced many companies to discover new strategies to produce natural flavors such as vanillin (Luziatelli et al. 2019).
Biotechnological approaches have been known for many years and they include more environment-friendly processes compared to chemical synthesis (Franco et al. 2017). Compounds produced by biotechnological routes are labeled as “natural” according to Europe (EC 1334/2008, EC 1223/2009, EC 872/2012) and US (US CFR 1990) regulations (Castro et al. 2021). Isoeugenol has been raised concern of researchers being convertible to vanillin in one-step besides to be cheap and commercially available (Wang et al. 2021). Unfortunately, majority of aromatic compounds has cytotoxic activity on biocatalyst cells (Priebe & Daugulis 2018). In recent years, biphasic system (organic/inorganic) has been preferred in bio-flavor studies to overcome this problem. In this method, a non-aqueous phase is used to sequester the substrate from aqueous solution that includes the cells. Low amount of substrate is continuously transferred between the phases from non-aqueous to aqueous (Priebe et al. 2018). It also provides a protection to the cells against product toxicity (Bicas et al. 2010). Using biphasic medium offers numerous advantages for biotransformation studies apart from prevention of toxicity. Dissolution of product in one of the phases ensures in situ removal and makes recovering process easier (Chreptowicz & Mierzejewska 2018). Losses depend on volatility can be decreased in biphasic system, especially working with monoterpenic constituents. Bioconversion yield may be raised by manipulating oxygen transfer rate in medium according to organic phase features (Bicas et al. 2010). It was investigated usage of biphasic system in various flavor production studies such as cinnamyl alcohol (Zhang et al. 2020), 2-phenylethanol (Chreptowicz & Mierzejewska 2018), R-(+)-α-terpineol (Bicas et al. 2010) and concluded that the method was very effective on raising catalytic performance of the cells. Unlike the other vanillin studies, Zhao et al. (2005) used a biphasic medium containing 60% (v/v) isoeugenol and achieved to produce 32.5 g L-1 vanillin which is the highest in literature, according to our knowledge.
A strong growth is expected in natural vanillin market with a compound annual growth rate (CAGR) of 7.4% from 2017 to 2025. In natural vanillin production, biotechnological approaches give an opportunity for producers (Luziatelli et al. 2019). Unfortunately, the production amounts remain very low due to substrate toxicity and hence, basic dispersion method is not applicable for industrial production. Thus, it is put emphasis on developing of current processes or constructing new alternative routes (Franco et al. 2017). Biphasic system has been come to the fore with high production rates as mentioned above. There is limited number of works for vanillin production in organic/inorganic bioconversion medium. Effects of temperature, pH and aeration in aqueous bioconversion medium were reported in our previous study (Karakaya & Yılmaztekin 2020). However, it was not possible to give results of biphasic (organic/inorganic) system within the same article due to limited scope. In this study, it was aimed to investigate biphasic medium usage for increasing vanillin production.
Natural Vanillin Production from Isoeugenol by Using Pseudomonas putida in Biphasic Bioconversion Medium
Hüseyin KARAKAYA Murat YILMAZTEKİN
Year 2022, Volume 28, Issue 3, 423–429
