It is generally considered that phytosterols
present in the diet, act as competitive inhibitors of cholesterol uptake in the
small intestine of humans (259).
Therefore, the blood cholesterol-lowering property made phytosterols to be in the
focus of significant efforts in academic and industrial labs (260).

Phytosterols as minor components are most
commonly obtained from vegetable oils (261).
Increasing the phytosterol content of oil seed is an important aim, because of
the existing facility of oil seed processing and the opportunity of delivering
bioactive phytosterols as a vegetable oil component without further processing (260).

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3-Hydroxyl-3-methyl glutary
Co-enzyme A reductase (HMGR) catalyzes the formation of mevalonic acid, a key
intermediate for triterpene synthesis (262)
Co-expression of HMGR gene from Hevea brasiliensis (rubber tree) lead to
an 11-fold increase in seed HMGR activity and increased the total phytosterol
content of tobacco seeds 2.5-fold to 3.25% of the total oil (261).
Phytosterol increases in seed have also been achieved through over-expression
of SMT1, the sterol C-24 methyltransferase type 1 at the entry point of
phytosterol biosynthesis (263).

Another approach to increase phytosterol
content in seeds was recently reported in the patent literature by McGonigle et
al. (264).
It consisted of suppression of certain pathways, such as saponin biosynthesis,
that compete with phytosterol biosynthesis for squalene 2,3 oxide (264).

In recent years, phytosterols from
microalgae are starting to attract more attention due to the diversity of
phytosterols in these species. The utilisation of microalgae for phytosterols
production offers an opportunity for finding novel phytosterols with potential
benefits to health or a mixture of molecules able to synergistically enhance
the bioactivity of a single phytosterol (12).

Methods for green extraction technology
of phytosterols include supercritical carbon dioxide extraction (SC-CO2).
Carbon dioxide has been used as the first choice solvent in more than 90% of
the supercritical fluid extraction of bioactive compounds from natural
resources, due to the benefits of safe, inexpensive, recyclable and being
non-hazardous to health and environment. This processing technology applies to
various sources and is considered to be an effective and environmentally
friendly technique for the separation of solvent-free phytosterols (12).

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