cofactor diffusion and in situ regeneration. Frutarom Ltd. has produced an enzyme
for the commercial production of a compound from synthetic ingredients which
gives the natural flavour of methyl mercaptan, found in all savoury vegetable or
meat-based flavours (BBSRC 2014). Another enzyme which is commercially available is Flavorzyme
® , manufactured by ‘Novo Nordisk Bioindustrials’, can be used
to obtain a meat-like flavour from defatted soybean meal (Dubal et al. 2008).
However, long and complicated steps for enzyme isolation and purification are
one of the disadvantages of these methods. Similarly, the differences in solubility
between reacting molecules are also a drawback of enzymatic processes which are
usually performed in aqueous phase (Sarma et al. 2014).
7.3.2 Microbial Methods
de novo synthesis of the flavour by microorganisms involves metabolism on
substrates such as carbohydrates (glucose and sucrose), fats and proteins by
microorganisms to form different and complex fragrant compounds (Dubal et al.
2008; Braga et al. 2018). The precursors usually used are fatty acids, amino acids
and terpenes (Hosoglu et al. 2018). These methods produce a mixture of several
aroma compounds, which are actually secondary metabolites produced due to
metabolic activities of the microorganisms. As regards the de novo synthesis, the
microorganisms through the enzymes such as lipases, proteases, nucleases and some
glycosidases transform carbon or nitrogen compounds into flavour compounds. The
first ‘de novo’ synthesis of an aroma compound (2-amino acetophenone) by Pseudomonas aeruginosa was reported by Omelianski (1923). Other species of Pseudomonas with striking odour properties were also identified such as aromaticus,
esterifaciens, odorus, odoratus, jragi or nobilis. The aroma formation was found
to be a ‘changeable character’ which was rapidly lost during ‘artificial cultivation’ of
the bacteria. The production of odour by a microbial culture has been explained due
to a single volatile, or due to an entire profile of volatiles, which are also concentration dependent in batch cultivation (Berger 1995). de novo methods can be practically applicable only when known starter cultures having good flavour potential are
used (Belin et al. 1992). These methods produce flavours in low concentrations. In
order to enhance production, genetic engineering techniques can be applied to de
novo processes, which involves expressing the genes from flavour producing
microorganisms into other microorganisms. Genes of plant and animal origins that
encode useful flavour molecules can also be expressed. However, the application of
genetically engineered de novo approaches is sensitive due to the complexity of
cellular potential and is also subjected to regulation in the host microorganism.
A limitation of de novo process is that the precursors which are usually added in
the medium may be inhibitory to the producer strains. Fed-batch processes can be
used to overcome substrate inhibition by slowly feeding the substrate and thereby
keeping the concentration of the compound below critical threshold values
(Carlquist et al. 2015). However, de novo synthesis is not very promising and
economically viable for industrial production as only trace amounts of flavours are
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T. Malik and S. Rawat
for the commercial production of a compound from synthetic ingredients which
gives the natural flavour of methyl mercaptan, found in all savoury vegetable or
meat-based flavours (BBSRC 2014). Another enzyme which is commercially available is Flavorzyme
® , manufactured by ‘Novo Nordisk Bioindustrials’, can be used
to obtain a meat-like flavour from defatted soybean meal (Dubal et al. 2008).
However, long and complicated steps for enzyme isolation and purification are
one of the disadvantages of these methods. Similarly, the differences in solubility
between reacting molecules are also a drawback of enzymatic processes which are
usually performed in aqueous phase (Sarma et al. 2014).
7.3.2 Microbial Methods
de novo synthesis of the flavour by microorganisms involves metabolism on
substrates such as carbohydrates (glucose and sucrose), fats and proteins by
microorganisms to form different and complex fragrant compounds (Dubal et al.
2008; Braga et al. 2018). The precursors usually used are fatty acids, amino acids
and terpenes (Hosoglu et al. 2018). These methods produce a mixture of several
aroma compounds, which are actually secondary metabolites produced due to
metabolic activities of the microorganisms. As regards the de novo synthesis, the
microorganisms through the enzymes such as lipases, proteases, nucleases and some
glycosidases transform carbon or nitrogen compounds into flavour compounds. The
first ‘de novo’ synthesis of an aroma compound (2-amino acetophenone) by Pseudomonas aeruginosa was reported by Omelianski (1923). Other species of Pseudomonas with striking odour properties were also identified such as aromaticus,
esterifaciens, odorus, odoratus, jragi or nobilis. The aroma formation was found
to be a ‘changeable character’ which was rapidly lost during ‘artificial cultivation’ of
the bacteria. The production of odour by a microbial culture has been explained due
to a single volatile, or due to an entire profile of volatiles, which are also concentration dependent in batch cultivation (Berger 1995). de novo methods can be practically applicable only when known starter cultures having good flavour potential are
used (Belin et al. 1992). These methods produce flavours in low concentrations. In
order to enhance production, genetic engineering techniques can be applied to de
novo processes, which involves expressing the genes from flavour producing
microorganisms into other microorganisms. Genes of plant and animal origins that
encode useful flavour molecules can also be expressed. However, the application of
genetically engineered de novo approaches is sensitive due to the complexity of
cellular potential and is also subjected to regulation in the host microorganism.
A limitation of de novo process is that the precursors which are usually added in
the medium may be inhibitory to the producer strains. Fed-batch processes can be
used to overcome substrate inhibition by slowly feeding the substrate and thereby
keeping the concentration of the compound below critical threshold values
(Carlquist et al. 2015). However, de novo synthesis is not very promising and
economically viable for industrial production as only trace amounts of flavours are
136
T. Malik and S. Rawat