Biotechnological production of vanillin from natural feedstocks
Author(s)
Di Matteo, Paola
Date Issued
February 17, 2011
Type
Doctoral Thesis
Abstract
This PhD research project was focused on innovative biotechnological production and recovery of vanillin and vanillin precursors. Nowadays, flavours cover over a quarter of the world market for food additives. Flavouring compounds are mainly produced via chemical synthesis or by extraction from natural materials. Flavours obtained by chemical synthesis of starting natural substances cannot legally be labelled as natural and the environmentally unfriendly production processes are subject to various problems such as lacks substrate selectivity, which may cause the formation of unwanted compounds thus reducing process efficiency and increasing downstream costs. On the other hand, the extraction processes from plants is often expensive because of the low concentrations of the molecule of interest in the raw material. Moreover the cost depends on uncontrollable factors such as plant diseases and weather conditions. The drawbacks of both methods and the increasing interest of consumers in natural product, reported in recent market survays, have led to the search for other strategies to produce natural flavours. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the most widely used flavoring in food and pharmaceutical industries. Chemically synthesized vanillin accounts nowadays for more than 99 % of the total market share. Extraction from vanilla beans is expensive and limited by plant supply, curing time and labour cost. Those factors make vanillin a promising target for biotechnological flavour production. As the Regulation (EC) no 1334/2008 of the European Parliament and of the Council of 16 December 2008 specify, vanillin produced in biotechnological processes starting from natural substrates can be classified as natural flavouring on condition that the natural starting material is specified. In recent years a large number of studies have been made on natural vanillin biosynthesis using microorganisms or isolated enzymes. However, these bioconversions are not yet economically feasible.
The high chemical activity and toxicity of vanillin cause low yield from ferulic acid. Moreover little vanillin was accumulated due to the higher degrading rate of this molecule than that of ferulic acid.
Biovanillin can be synthesized using cells or enzymes starting from different natural compounds, such as ferulic acid, eugenol or capsaicin. The latter, ((6E)-N-(4-hydroxi-3-metoxibenzil)-8-metil-6-nonenamide) is the pungent compound in chili pepper related plants of the Capsicum family. It can be hydrolyzed to vanillylamine, (4-hydroxy-3-methoxybenzylamine), a natural precursor of vanillin, by cleavage of its amine bond using specific microbial acylases. The aims of the thesis were: to enhance vanillin production
2
from resting cells of E. coli engineering strains, starting from ferulic acid and by using XAD-4® resin and a new two/phase system of agarose gels and ferulic acid for the controlled release of the substrate in the bioconversion medium (1), to evaluate different strategies for enhancing the production of the capsaicin acylase from Streptomyces mobaraensis DSM40847 strain (2), to identify optimal conditions for capsaicin hydrolysis by using the acylase from Streptomyces mobaraensis DSM40847 strain (3), to develop efficient procedures for the recovery of vanillin from diluted aqueous solutions (4).
Experiments carried out using whole cells or crude enzyme preparations demonstrated that: (a) the two phase system of agarose gel and ferulic acid developed in this work was compatible with the bioconversion and it permitted to reduce the toxic effect of the ferulic acid; (b) selective recovery of the product, using macroporous resins in an off-line process, enhances the biological conversion of ferulic acid to vanillin using resting cells of Escherichia coli engineering strains; (c) thermal pre-treatment at 55°C of crude PVA acylase from Streptomyces mobaraensis DSM40847 improves the conversion of capsaicin to vanillylamine; (d) liquid/liquid extraction with n-butyl acetate allowed high recovery and high selectivity and supplied the best result in the recovery of vanillin from aqueous solutions.
Additional information
Dottorato di ricerca in Biotecnologia degli alimenti
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