Design and development of environmental friendly products and processes for food safety and biosensoring
Author(s)
Bozzini, Tiziana
Date Issued
May 30, 2014
Type
Doctoral Thesis
Abstract
This work concerns topics of food safety. With the aim to preserve the quality, nutritional
value and chemical composition of foods, we studied the synthesis of novel antioxidant
compounds, and prepared simple scaffolds based on the immobilization of enzymes on
epoxy resins and carbon nanotubes (CNTs) for synthetic and biosensoring purposes. To
achieve this goal we synthesized antioxidants characterized by two pharmacophores, the
catechol moiety and a long carbon side-chain, able to give important biological properties
at the new molecules. Our synthetic strategy was based on two enzymes, lipase from
Candida antarctica and tyrosinase from Agaricus bisporus. The use of enzymes as
biocatalysts has received an increasing interest for the synthesis of organic compounds,
because they have a high catalytic efficiency and selectivity, that allow the specific
recognition of substrates and products, even in complex mixtures. Furthermore, these
biocatalysts generally work under mild conditions of reaction: pH near to neutrality,
temperatures below 100°C and at atmospheric pressure (in few words they are
environmental friendliness). As a general procedure, a large panel of acid phenols was
esterified by lipase from Candida antarctica to introduce a carbon side-chain on the
aromatic ring able to increase the solubility in the lipid cellular compartments. The esters
were then directly oxidized by tyrosinase from Agaricus bisporus, to give catechols.
Subsequently, the two enzymes were used in cascade (in only one step) to give the desired
products in a more shorter multi-enzimatic pathway, reducing time and costs of reaction.
Since there are some limits in the use of enzymes (i.e. because of their low stability, high
cost of production, possible inhibition by impurities, difficulty to be recovered from the
mixtures, etc.), we also applied some immobilization procedures. The immobilization
techniques offer the possibility to separate the enzyme from the product, and reuse it in
continuous production processes. For this reasons, tyrosinase was immobilized on the
epoxidic resin Eupergit-CM, and then the new biocatalyst was covered with
polyelectrolytes by the Layer-by-Layer method. The novel heterogeneous biocatalysts
were used for the synthesis of catechols and fully characterized for their storage stability
and the possibility to reuse in novel reactions. All new products were evaluated for their
antioxidant activity in vitro through DPPH assay. Data showed that the antioxidant activity
was present only with the catechol function, and increased by increasing the length of
lipophilic side chain. Since antioxidant compounds are not important only for food safety,
but also to the human health, the new products were also evaluated for the antiviral
activity against influenza A virus. New products show two important activities: antioxidant
and antiviral properties.
These results were applied to another important molecules, L-DOPA, that have an
important role in the mechanism of enzymatic browning of foods and a considerable effect
on several diseases of the central nervous system, mainly caused by oxidative damage. So
new bioactive 3,4-dihydroxyphenylalanine peptidomimetics were synthesized with a
similar approach. In particular, N-BOC protected amino acids were used as a nucleophile
to study the addition on a reactive DOPA-quinone intermediate, generated by in situ
oxidation of L-DOPA. Finally, tyrosinase was immobilized on multi-wall carbon
nanotubes (MW-CNTs) and used as catalyst for the oxidation of phenols to corresponding
catechols in organic solvent. The new catalysts performed the oxidation with a similar
selectivity, but a minor amount of enzyme was required respect to previously described
systems based on epoxy resins. This means a significant advantage in terms of cost
savings. The new biocatalyst immobilized on CNTs could be used also as biosensor to
detect the presence of phenols and catechols in foods.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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