Natural flavourings from biotechnology for foods and beverages - Natural Food Additives, Ingredients and Flavourings - page 249

4000 per kg and has a market of 50 tons/year (Feron and Waché 2006). The term
'natural' can be applied both in the European Union (EU) and in the United States
when the product has been derived from a natural raw material via biological (e.g.
enzymes or whole cells) and/or mild processing tools (e.g. extraction or distillation)
(Lesage-Meessen et al. 1996). Thus, vanillin is the subject of much attention in
the EU at the moment because the new regulations exclude certain natural sources
of this compound. These sources use processes derived from pressure reactions
above 120°C (derived from eugenol). Nowadays, new routes are being explored
to produce vanillin using abundant precursors. Vanillylamine can be obtained
from the precursor compound capsaicin (8-methyl-N-vanillyl-6-nonenamide), the
pungent principle of hot red pepper, by the cleavage of its amide bond. Thus, the
enzyme fl avoprotein vanillyl alcohol oxidase from Penicillium simplicissimum
could act on a wide range of phenolic compounds and convert both creosol and
vanillylamine to vanillin with high yield. Heuvel et al. (2001) described two
enzymatic routes to produce vanillin: (i) the fl avoprotein, vanillyl alcohol oxidase
(VAO), which acts on a wide range of phenolic compounds and converts both
creosol and vanillylamine to vanillin with high yield; and (ii) the VAO mediated
conversion of creosol proceeds via a two-step process in which the initially
formed vanillyl alcohol is further oxidized to vanillin.
Neither de novo routes in plant cell cultures of vanilla nor those in bacteria or
fungi afford anything like acceptable yields. The precursor approach holds more
promise. Several starting materials appear to be suitable including lignin, eugenol,
ferulic acid, curcumin and benzoe siam resin (Benz and Muheim 1996). Turnover
rates of less than 30% and production levels below 1 g L −1 have been reported.
Again, the toxicity of both the precursor and the product to microorganisms cells
(vanillin is not toxic to humans being listed as GRAS (Generally Recognized As
Safe) by the US Flavor and Extract Manufacturers Association (FEMA)), as well
as product degradation in the course of fermentation, prevented a better yield (Zorn
et al. 2003). Although the highest yields of biotechnological vanillin are related to
patented biotransformation/bioconversion processes, with precursors such as
ferulic acid and eugenol (Dausgh and Pastore 2005), some processes obtained this
compound by de novo means, mainly from glucose by recombinant Escherichia
coli (Li and Frost 1998) and Schizosaccharomyces pombe (Hansen et al. 2009).
A large number of patents and papers describe different bioprocesses to obtain
vanillin by biotechnological means. The highest yields reported for
biotechnologically produced natural vanillin refer to patent processes: the
bioconversion of ferulic acid into vanillin by strains of Amycolatopsis sp. or
Streptomyces setonii in a ten-liter bioreactor. The fi nal yields were 11.5 g L −1
(Rabenhorst and Hopp 2000) and 13.9 g L −1 (Muheim et al. 1991) respectively. In
fact, the microbial transformation of ferulic acid is recognized as the most
attractive and promising alternative source of natural vanillin (Bicas et al. 2010c).
Figure 11.4 describes the biosynthetic pathways of vanilin, vanillyl alcohol and
vanillic acid production from caffeic acid.
For more details about the bioproduction of vanillin, see Walton et al. (2000),
Walton et al. (2003), and Daugsch and Pastore (2005).

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