Bt Research 2015, Vol.6, No.4, 1-12
8
Another important factor in the assessment of
environmental performance and safety, in addition to
the physiological toxicity, is related to the stability of
the thuringiensins (Zhou et al., 2013). Initially, several
authors have reported that these exotoxins were quite
persistent in the environment (Beebee and Korner
1972; Benz 1966; WHO 1999). Hitchings (1967)
reported that thuringiensins are not degraded by
exposure to UV radiation. However, (Zhou et al., 2013)
recently reported that this exotoxin is unstable in
aqueous solution and can be degraded in non-toxic
compounds, being that this process can be reinforced
by modification of biotic, physical or chemical factors,
particularly with respect to pH and temperature.
According to these authors, thuringiensins are highly
unstable under simulated environmental conditions,
have half-life around 2.72 to 16.19 days in the aqueous
phase (significantly shorter than other common
environmental pollutants) and can be 99% degraded
without the presence of other microorganisms. Thus,
under natural environmental conditions that contain
different types of microorganisms, the degradation
process can be accelerated. This data can decrease the
concerns about the security issues of this toxin.
8 Procedures for the Detection of Thuringiensins
The first purification methods of thuringiensins
involved centrifuging processes (Barjac et al., 1966);
precipitation with CaCl
2
(Kim et al., 1970); micellar-
enhanced ultrafiltration with surfactant cetylpyridinium
chloride (CPC) (Tsun et al., 1999); fixation by calcium
silicate and sodium phosphate dibasic for dissociation
in the middle of fermentation, followed by high-per-
formance liquid chromatography (HPLC) and
electrodialysis to remove excess salts from solution
(Tzeng et al., 2001); or precipitation for acetone and
acetonitrile added to the supernatant (Gohar et al.,
2001). Methods involving ultrafiltration have several
drawbacks, including the high cost, low efficiency and
extended duration. The method proposed by (Gohar et
al., 2001) is able to detect very low levels of thuringiensins
(0.3 μg/mL) and thus is the most widely used.
The most traditional method for detection of
thuringiensins is based on toxicity bioassays (Gohar
and Perchat 2001; Hernândez et al., 2001). As
Musca
domestica
larvae are very susceptible to such exotoxins
and do not develop into normal adults after exposure
to the toxin, bioassays with these organisms may be
used for the identification of these exotoxins (Bishop
and Robinson 2014; Cantwell et al., 1964; Mac Innes
and Bouwer 2009; Mechalas and Beyer 1963). Several
authors propose high-performance liquid chromatography
(HPLC) assays as a rapid method to detect and
quantify thuringiensins. This method presents itself as
an alternative to bioassays, which are time consuming
(up to 9 days), enough variables, non-specific and can
estimate inaccurate potencies due to impurities in the
samples (Bubenschikova et al., 1983; Campbell et al.,
1987; Rodríguez et al., 2003; Liu et al., 2010). The
limit of detection of thuringiensins by HPLC ranges
from 0.1 to 10 μg/mL (De Rijk et al., 2013). However,
the HPLC method does not evaluate the direct toxicity,
can provide false negatives and requires expensive
equipment (Rodríguez et al., 2001; Mac Innes e
Bouwer 2009). In turn the ELISA method is more
sensitive than HPLC, can be used to quantify and
quantitate the thuringiensins, with a detection limit of
0.1 ng/mL. (Liu et al., 2010) used the method of liquid
chromatography mass spectrometry (LC-MS) for the
qualitative confirmation of exotoxins, with the aim of
detecting the presence of thuringiensins, even at very
low concentrations. (Sauka et al., 2014) propose a
method of Polymerase Chain Reaction (PCR) for the
rapid prediction of the production of thuringiensins of
type I by detection of the
Thue
gene, strongly
associated with the synthesis of this exotoxin type.
(Espinasse et al., 2002b), to report a strong correlation
between production and the presence of Cry1B gene
with the synthesis of thuringiensins, also suggest that
a PCR reaction could allow indirect detection of the
toxin. However, as the secretion of this exotoxin
depends on the time of growth, the use of this
technique does not seem to be reliable about the point
of view of these authors (Argôlo-Filho et al., 2014). In
addition, other methods have been developed, including
ion exchange chromatography, spectrophotometry and
micellar electrokinetic capillary chromatography,
quantification a limit of 0.028 mg/kg (Levinson et al.,
1990; Campbell et al., 1987; De Rijk et al., 2013). The
production of this toxin can still be evaluated in
laboratory tests using rodent models (Burges 1981;
Pinto et al., 2010).
Despite advances in research about thuringiensins of
type I, detailed studies are needed on the structure,
characteristics and mode of action of thuringiensins of
type II. More specific tests with several non-target
