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(Odoux, 2000). Consequently, vanilla pods treated with glucosidases from external sources yielded vanillin in high concentrations than the pods cured normally (Dignum et al., 2001; Ruiz‐Teran et al., 2001). Mane and Zucca (1993) have shown that enzymatic treatment with 40–400 units of β‐glucosidase per gram of green vanilla pods is effective for better production of vanillin. The traditional curing process provides only 2% of vanillin yield because uncured green beans contain 10–15% of glucovanillin. Thus, exogenous enzyme preparations containing cellulase, pectinase, and β‐glucosidase were added to increase the yield. Nevertheless, with the addition of exogenous enzymes on green pods 4.25–7.00% of vanillin yield had been achieved (Perera and Owen, 2010). Recently, Naidu et al. (2012) has demonstrated that treatment of tea leaf enzyme extracts (TLEE) with vanilla flavor precursors produced higher content of vanillin (4.2%) with superior flavor quality than Viscozyme‐treated vanilla extracts (2.4%).
The escalating demand for the natural vanillin in the world due to low yields from natural vanilla pods, biotransformation of other plant‐derived materials like ferulic acid, stilbenes, lignin, and eugenol through enzymatic hydrolysis are being developed to produce high‐quality vanillin. Ferulic acid, a cheap raw material found abundantly in the plant biomass (Zheng et al., 2007), is one of the most extensively investigated substrate to produce vanillin through biotransformation. The feruloyl‐CoA synthetase (Fcs) that degrades ferulic acid and enoyl‐CoA hydratase/aldolase (Ech) that produces vanillin were characterized in many microbial sources. For example, the enzyme preparations from the recombinant Escherichia coli expressed with Ech and Fcs genes from Amycolatopsis sp. strain HR167 and Streptomyces sp. strain V‐1 resulted in successful conversion of ferulic acid to vanillin (Achterholt et al., 2000; Yang et al., 2013). Van den Heuvel et al. (2001) used vanillyl alcohol oxidase (VAO) obtained from Penicillum simplicissimum, which is a flavoenzyme catalyzes the conversion of vanillylamine (the active principle of pungency in chili peppers produced as an intermediate during capsaicin biosynthesis) and creosol (a carbonaceous material obtained by the pyrolysis of wood and distillation of coal tar) to vanillin with high yield. Similarly, eugenol oxidase (EUGO) is a flavoenzyme produced by Rhodococcus sp. RHA1 that catalyzes conversion of vanillyl alcohol to vanillin (Jin et al., 2007). Garcia‐Bofill et al. (2019) immobilized EUGO on different supports such as MANA‐agarose, Epoxy‐agarose, and Purolite 8204F to improve its stability in oxidizing vanillyl alcohol for enhanced production of vanillin, which resulted in 2.9 g l/1 H of vanillin.
Lipoxygenase (LOX), a class of iron‐containing dioxygenase present in high concentrations in soybean, is well known for catalyzing the hydroperoxidation of polyunsaturated fatty acids and esters. LOX has also been reported to catalyze the oxidative cleavage of isoeugenol to vanillin (Li et al., 2005), which holds potential as a promising route for enzymatic synthesis of vanillin. Liu et al. (2020) used soybean LOX as the catalyst for vanillin synthesis from isoeugenol through addition of denaturants (urea and guanidine) and chelators (EDTA), which were effective in improving yield of vanillin by up to 133% and 406%, respectively than control. Overall, enzymatic production of vanillin seems attractive, though their performance in bioreactors or fermenters in terms of stability and activity that remains elusive.
2.3.2 Microbial Biotransformation of Ferulic Acid to Vanillin
Microbial transformation of phenolic compounds into vanillin is the most attractive alternate for natural vanillin. Many microbes are capable of producing vanillin and vanillic acid by utilizing phenolic substrates such as ferulic acid, eugenol, and isoeugenol (Figure 2.2). Among them, ferulic acid has been widely used as a sole carbon source for vanillin production. Pseudomonas fluorescens is one of the most predominantly used organisms for such biotransformation. Under optimized environmental conditions, P. fluorescence BF13 is capable converting 95% of ferulic acid to vanillic acid in five hours (Barghini et al., 1998). Likewise, a mutant of P. fluorescens FE2 is shown to produce 95% of vanillic acid from ferulic acid in 24 hours (Andreoni et al., 1995). Next to Pseudomonas sp., Lactobacillus sp. is considered to be effective producer of vanillin as it has phenolic transformation properties. A group of Lactobacillus strains are found to produce significant amount of vanillin from ferulic acid supplemented rice bran medium (Kaur et al., 2013). Streptomyces setonii and Amycolatopsis sp. have been reported to produce notable amount of vanillin under optimized pH (Rabenhorst and Hopp, 2000; Gunnarsson and Palmqvist, 2006). Likewise, Delftia acidivorans also has been reported as potent bacteria to transform ferulic acid to vanillin efficiently (Plaggenborg et al., 2001). The second most commonly used carbon source in biotransformation of vanillin is eugenol and isoeugenol, which are essential oil components extracted from clove. The degradation pathway of eugenol in certain bacteria like Corynebacterium sp. (Tadasa, 1977), Arthrobacter globiformis (Rabenhorst, 1996), Pseudomonas sp. (Tadasas and Kayahara, 1983) has resulted in producing vanillin, vanillic acid, and other phenolic compounds. Arthrobacter sp. TA13, Pseudomonas putida JYR‐1, and Bacillus subtilis B2 are reported to produce vanillin from isoeugenol (Shimoni et al., 2000, 2003; Ryu et al., 2005). Bacillus fusiformis has been reported to produce 8 g/l of vanillin from 50 g/l of isoeugenol with the addition of HD‐8 resin to prevent inhibition by product (Zhao et al., 2006). Further, Bacillus pumilus S‐1 is shown to transform isoeugenol to vanillin with higher efficiency with the yield of 3.75 g/l of vanillin from 10 g/l of substrate in 150 hours (Hua et al., 2007). Bacillus licheniformis SHL‐1 has been reported to produce 494 mg/l of vanillin from 1 g/l of ferulic acid within 45 hours (Ashengroph et al., 2012). Other than bacteria, Aspergillus niger is also most frequently used to transform ferulic acid to vanillic acid, which is further converted to vanillin by Pycnoporus cinnabarinus or Phanerochaete chrysosporium. The yield of vanillin by this two‐step process is reported to be 1.1 g/l after 54 hours of fermentation (Stentelaire et al., 2000). Sporotrichum thermophile, a thermophilic fungus has also been reported to produce 4798 mg/l within 20 hours of fermentation using ferulic acid as a substrate (Topakas et al., 2003). On the other hand, vanillin production by fungal bioconversion has many disadvantages, such as lysis of mycelium, extremely viscous broths, uncontrolled fragmentation, and unfavorable pellet formation, which hinder conventional production processes and increase the price of the downstream process. Apart from these, a list of microbes involved in biotransformation of vanillin is listed in Table 2.1. Besides having good yield, biotransformation process has several disadvantages like formation of undesired by‐products, expensive downstream processing, unproductive metabolic flow, cytotoxicity of precursors used (Gallage and Møller, 2015), and the difficulty in process optimization of the microbes used. In order to overcome these problems and to meet the market demand, biotechnological approaches including metabolic and genetic engineering are being employed and these methods have gained much importance nowadays because of higher yield of vanillin.
Figure 2.2 Biotransformation pathways of vanillin in microbes using various substrates.
Table 2.1 List of microorganisms capable of producing vanillin from various substrates.
| Organism | Substrate | References |
|---|---|---|
| Gram‐positive bacteria | ||
| Amycolatopsis sp. HR167 | Eugenol | Overhage et al. (2006) |
| Rhodococcus opacus PD630 | Eugenol |
Plaggenborg et |