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Determination of Aflatoxins | J St | YouTubeToText
YouTube Transcript: Determination of Aflatoxins
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Video Summary
Summary
Core Theme
This content details a validated analytical method for determining aflatoxin B1 in feedstuffs, emphasizing accuracy, reproducibility, and safety precautions for handling mycotoxins.
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this video is complementary to the
written method it concentrates on
critical aspects of the methods
important in achieving accurate and
reproducible results for further details
please refer to the printed description
of the method
the latter is based on the most
up-to-date techniques currently
available and was developed by the
German VDL UFA the method has not been
previously applied in this form to feedstuff
regarding the procedures described in
this video all normal laboratory safety
precautions must be taken additionally
special precautions must be made in
relation to the handling of mycotoxins
these include the wearing of gloves and
glasses the handling of spillages and
the disposal of contaminated waste these
precautions are also listed in the
a wide variety of food or feet stuff
such as cereals maize ground nuts
cottonseed and so on may be contaminated
by moles that produce a full range of
chemicals named mycotoxins among these
aflatoxins are toxic metabolites of
Aspergillus flavors and a specialist
parasitic as' and have been found in
many food products
moreover aflatoxins are important in
animal health because they may occur in
animal feed stuff when cattle are given
contaminated feed aflatoxins can find
their way into dairy foods some
mycotoxins present a potential hazard to
human or animal health even when present
at very low concentrations their levels
in food and animal feed stuff are
controlled by regulations in more than
validated methods for the analysis of
mycotoxins are necessary in order to
apply these controls and to set
standards for trading purposes therefore
the food analysis unit of the European
Commission's joint research centre has
undertaken the initiative to validate a
suitable analytical method for the
determination of aflatoxin b1 in
feedstuff naturally contaminated
feedstuff material with various
concentrations of aflatoxin b1 in low
ppb micro kilogram ranges are the basis
for the validation study the feedstuff
contains also commonly used ingredients
which might interfere with the detection
of aflatoxins the analysis of aflatoxin
b1 is based on the extraction from the
feedstuff followed by an immuno affinity
column cleanup step for sample
preparation and further analysis by
high-performance liquid chromatography
when the feedstuff sample consists of
compound material or grain an
appropriate milling has to be carried
out prior to extraction ground feedstuff
material may be extracted and analyzed
directly a ground feedstuff sample of
about 50 grams is weighed out in an
Erlenmeyer flask it is very important to
record the exact weight the feed staff
is then diluted in 250 milliliters of an
acetone water mixture shaken intensively
by hand for a few seconds to achieve a
homogenous suspension and then shaken
mechanically at moderate speed for 30 minutes
filtration is then carried out through
folded filter paper an aliquot of five
milliliters is taken from the filtrate
and diluted to 100 millilitres in a
volumetric flask by using phosphate
buffered saline or water according to
the specifications for the immuno
if the solution isn't clear it must be
refill turd through a glass fiber filter
prior to the immuno affinity clean up if
it's clear it's ready for being
transferred on to the immuno affinity
column no specific manufacturers columns
are recommended however minimum
performance criteria are specified for
this method if the columns are not
guaranteed to meet these criteria they
must be tested in the laboratory the
immuno affinity column contains
antibodies which are specific to the
target mycotoxin the antibodies are
bound to the packing material in the
column they are selectively bind the
target mycotoxin and remove it from the
extract when the sample is passed down
the column the right flow rate is
important in order to bond all a flow
toxin b1 to the antibody the column is
then washed to remove any other
potentially interfering substances which
may still be present finally the bond
between antibody and mycotoxin is broken
by Ellucian with the solvent and
aflatoxin b1 in a very pure form is collected
to collect pure aflatoxin b1 an aliquot
of exactly 50 milliliters of the diluted
sample extract is applied on the immuno
affinity column the sample is poured
into a syringe barrel attached to the
top of the column the solution may be
passed through the immuno affinity
column by gravity or a slight vacuum if
the vacuum is applied the column should
not be allowed to dry out the flow rate
should not exceed three to five
milliliters per minute so the total time
for loading onto the column will not be
less than 17 minutes after loading the
column is washed twice with 10
milliliters of water and then dried by
the aflatoxin b1 is eluted from the
column in two stages firstly North point
seven five milliliters of methanol is
applied to the column and allowed to
pass by gravity the Lu it is collected
in a calibrated five milliliters
volumetric flask after one minute
another portion of one millimeter of
methanol is added in this case the Eliot
is collected by applying pressure or
passing air through the column after
most of the solvent has passed through
this allows to collect most of the
applied solvent with the aflatoxin b1
from the immuno affinity column the
volumetric flask is made up to volume
with water and is ready for injection
into the HPLC system as methanol and
water contract while shaken adjust the
level after shaking in case the solution
is cloudy it should be passed through a
disposal filter unit if it's clear it's
200 microliters of the solution are
injected into the HPLC system the method
requires post column bromination of the
aflatoxin b1 prior to fluorescence
detection bromination can be carried out
either using Pb Pb reagent or using
electro chemically generated bromine the
experimental details for both approaches
are set out in the written method and
calibration curve should be established
by injecting solutions of the aflatoxin
b1 standards this curve which should be
linear is employed to determine the
concentration of aflatoxin b1 after HPLC
analysis the extracts from all feedstuff
samples should show a well resolved
aflatoxin b1 peak free from other
extracted components this is a typical
chromatogram from naturally contaminated
material showing a clearly discernible
fully automated systems such as the ASP
EC system can be used as well all
techniques can be applied provided the
analytical protocol is not modified in
terms of volumes of reagents or flow
covered and allowed to stand for 60
finally a 50 microliters of the stop
solution is added to each well the micro
plate is now ready for the measurement
of the color intensity the micro plate
is placed into a microplate reader a
photometric instrument designed to
measure the solutions the intensity of
the color of the solution each well is
measured automatically at 440 nanometer
or 405 nano meter for this application
the microplate reader uses air as reference
finally the printout of the microplate
reader and the template with the
information about the samples are used
to evaluate the results for further
details please refer to the printed
method description you
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