Note: Descriptions are shown in the official language in which they were submitted.
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This invention is directed to an analytical
test apparatus for a simple, rapid and reliable colorimetric
determination of ~lucose in compositions which contain
inhibitors of the desired colour development due to glucose.
The invention is particularly useful for the determination
of glucosinolates in oilseeds and oilseed meals, the
glucosinolates being enzymatically hydrolyzed to glucose
before assay. A kit including the test apparatus for
determinations in the field, is described.
Background and Prior Art
The determination of glucose is widely practiced
since this is the sugar which diabetics must monitor in their
body fluids. Glucose-specific test papers have been developed
; and are on the market. One commonly used test paper contains
I two enzymes (glucose oxidase and peroxidase~, the chromogen
o-tolidine, and a yellow dye. When an aqueous solution of
glucose comes in contact with this paper, hydrogen peroxide
` is produced by the action of glucose oxidase on glucose. The
resulting hydrogen peroxide in the presence of o-tolidine
and peroxidase forms a blue complex. Against the background
- of the yellow dye, the test paper appears green (the higher
the glucose concentration the more intense the green colour)
- and a colorimetric determination can be made. Inhibitors
of these colour-forming reactions are not normally encountered
in body fluids but may be encountered in other systems.
Glucosinolates are a family of sulfur-containin~
compounds, at least nine of which occur in the seed of
rapeseed (Brassica napus L. and Brassica campestris L.) and
other oilseeds such as mustard and crambe. They are
` 30 undesirable compounds because their hydrolysis products,
isothiocyanates, thiocyanates, nitriles and epithionitxiles
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(aglycones) cause metabolic upsets in non-ruminant animals.
Their presence in rapeseed is a major constraint on more
:intensive and widespread use of rapeseed meal in animal feeds
and potentially could limit the use of protein isolates or
concentrates for human consumption. Plant breeders, therefore,
have concentrated efforts on developing varieties reduced or
free of glucosinolates. The recent introduction in Canada of
new varieties of rapeseed such a~ Tower, Regent, Altex and
Candle with substantially reduced levels of glucosinolates,
has created a preference for low glucosinolate-containing
seed and meal in both the domestic and international markets. ~-
However, these markets can only be served if the new low
glucosinolate-containing seed and meal is kept separate from
high glucosinolate-containing seed and meal throughout the
production, processing, transportation and marketing system.
This is difficult because high and low glucosinolate-
containing seed and meal are visually indistinguishable.
Production and marketing would be greatly facilitated if
there were a better method available which was suitable for
use at all points in the production and marketing system to
distinguish low glucosinolate rapeseed or meal.
Analytical methods have been devised for
determining levels of glucosinolates by measuring inorganic
sulphate (released on hydrolysis) or the other aglycones. ~ -
However, since the procedures for these quantitative analyses
are time-consuming, technically demanding, and require
expensive chromatographic and spectrophotometric equipment,
they are of limited usefulness in commercial trade for
segregating seed according to glucosinolate content, and their
application has been confined to selection in plant breeding
and to control of pedigree seed lots.
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The first to demonstrate that glucose test
papers could be used to determine the approximate glucosino-
late level in rapeseed was K.A. Lein (Z. Pflanzenzuecht 63,
137-154, 1970). In 1972, Bjorkman reported the use of
glucose oxidase, peroxidase and the chromogen o-dianisidine
to colorimetrically estimate in solution the quantity of
purified glucosinolates from rapeseed extracts. However,
when such colorimetric analyses were applied to crude extracts
of glucosinolate-containing Cruci~erae including rapeseed,
interfering substances were found to inhibit colour develop-
ment (Van Etten et al, J. Agric. Food Chem. 22, 483-487, 1974).
These workers found that the inhibition could be overcome by
treating the extracts with charcoal before colorimetric
analysis or, when using glucose test paper, by separating
the inhibiting substances from glucose in an aqueous seed
extract through capillary action up a strip of test paper.
For rapid screening with test paper, Van Etten et al considered
the capillary separation up the test paper the most satis-
factory.
; 20 ~ However, for enhanced sensitivity and
reproducibility when rapeseed extrac's were being assayed,
; McGregor and Downey (McGregor, D.I. and Downey, R.K., A Rapid
and Simple Assay for Identifying Low Glucosinolate Rapeseed,
Can. J. Plant Sci. 55, 191-196, 1975) found that the addition
of charcoal was necessary. In the absence of charcoal, color
development of the glucose test paper with crude rapeseed
^' extracts was variable and mottled such that quantitative
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analysis at best was dif~icult if not impossible. The
controlled addition of activated carbon powder, resulted in
uniform color development over the whole test paper strip
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facilitating quantitative analysis. Color development
was enhanced, improving sensitivity so that as little as 10%
by weight contamination of seed with high glucosinolate
content/ in seed of low glucosinolate content,could be
detected. This test procedure of McGregor and Downey as
presently used to identify low glucosinolate rapeseed is
simple but somewhat "messy" and slower than desired. It would
be difficult to carry out in many field situations. To date
use of this assay (McGregor and Downey) has been limited to
analytical labs and crushing plants where facilities for
handling and disposing of the activated carbon and for
cleaning mortar and pestle, etc. are available.
~ or other analyses, various test devices,
laminates, sticks, etc. have been developed to simplify and
speed up the procedure. Typical prior art devices are
described in United States Patents: 3,011,874; 3,993,451;
4,061,468; 4,065,263; and 4,094,647. To applicant's
knowledge, no such devices have dealt with the problem of
inhibitors in a colorimetric glucose (or glucosinolate) assay.
Summar~ of the Invention
The present invention is primarily an
analytical test apparatus suitable for the colorimetric assay
of glucose in a liquid composition which also contains
inhibitors of the colour development, comprising:
(i) a light-coloured inert solid backing having a sample-
` receiving zone (a) spaced apart from an adsorbent zone
(b) and test zone (c),
(ii) a wick able to transport the liquid by capillary action,
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extending from zone (a) to zone (b) on said backing,
.
(iii) a selected adsorbent at zone (b) in capillary contact
with the wick, and
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(iv) glucose test paper at test zone (c) on said backing
in capillary contact with said adsorbent at a location
remote from the wick contact;
the adsorbent being selected to adsorb said inhibitors in
preference to glucose; the amount of adsorbent traversed
between the wick and the test paper being just sufficient to
adsorb the substances which inhibit colour development due to
glucose. The test apparatus is most suitably in the form of
a stick, strip, tape or sheet.
The invention includes a kit for the colori
metric assay of glucose in a composition derived frorn seeds
which contain inhibitors of the colour development, comprising:
~a) the test apparatus including appropriate colour
standards for the glucose test tape present,
(b) means to contain seed sample during crushing, and
(c) means to measure and contain both seed sample and water.
T~e kit optionally may include low~ and/or high-glucosinolate
seed standards, myrosinase for glucosinolate hydrolysis, or
; seed crushing means.
Description of Drawing
The single drawing is a front and side view
of a preferred test stick as described in de~ail in the
example below.
Detailed Description
` The backing for the test apparatus can be
chosen from inter alia sheet, film, stick or tube of plastics,
:, ~
; coated cellulosics (wood, paper, etc) or metal foils. The
backing should be inert, substantially non-absorbent and of
a light colour not interfering with the colour reading at
least in the test zone. One backing found very convenient is
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a photograph mounting sheet having sticky adhesive layers
covered on both surfaces by removable paper sheets.
The wick can be made of various porous
absorbent papers or fabrics able to conduct liquid by
capillary action. Cellulosic wicks in paper form are usually
inexpensive and readily available. However, other natural or
synthetic fibrous or particulate materials, or sponge material
could be used to form the wick.
- The adsorbent can be any high surface area
material preferentially a~le to adsorb and hold up the
interfering substance or substances. Activated carbon has
been found very suitable. Glucose itself will be slowly
adsorbed on activated carbon so that the amount of carbon
and the time of exposure should be limited to avoid readings
which are too low. It was found important in developing a
reasonably accurate test apparatus to determine the amount
of liquid that would soak up into the glucose test paper and
then incorporate into the apparatus upstream of the test
paper an amount of adsorbent to give an approximate ratio of
: .
; 20 adsorbent to liquid found necessary for an accurate assay.
This ratio of adsorbent to liquid (where the li~uid is about
0.02 to 0.2% glucose) has been determined to be about 1 mg
: .
carbon per 17.8 microliters or about 0.6 - 0.7 g per 12.5 ml.
It has been found convenient to form a layer of adsorbent on
a thin support, the thickness of this layex being controlled
to give the desired amount of adsorbent over an area the size
of the adsorbent zone. The support can be adhered readily
to the backing in the appropriate location.
A permeable binder may be added to the adsorbent
to aid in forming the layer just mentioned. A suitable
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binder is gypsum (plaster of paris). Other additives such
as chromatographic substrate powder may be added to the
adsorbent to aid in capillary transport or selective adsorption
in the layer.
The glucose test paper can be any available for
glucose determinations, having a good colour response for
amounts of glucose ranging from 0.02 to 0.2% wt./vol~ The
absorbent action or capillary action of the test paper should
be good in order to draw sufficient liquid composition for an
accurate determination. A suitable test paper would be that
impregnated with glucose oxidase and peroxidase, a chromogen
such as o-tolidine and a yellow dye such as F.D.C. yellow
No. 5.
The adsorbent layer is delicate and preferably
should be protected by an impervious covering such as a
I plastic film or tape. This protective covering desirably
will extend upstream to cover the wick between zones (a) and
(b), in which case the edge of the covering will serve to
define the limit of the sample-receiving zone (a). A pro- `
,
~ 20 tective covering may also be applied downstream of the test ~
~ , .
zone (c) to help delineate this zone, and if wick material
extends beyond the test paper - to protect this wick. Black
plastic tape has been found very suitable as a protective
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~, impervious covering, with strong contrast to the test paper,
wick, and backing.
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Example
A prototype in the form of an analysis stick
was constructed using photo print mounting backing consisting
of a white sticky center (see drawing at a) which was covered
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with two adhering layers of brown paper (b and h). The
brown paper was easily removed to expose a sticky surface.
Only one stic~y surface was used to construct the stick, the
other being le~t covered with brown paper. (If desired, the
test stick could be built on both sides to give a second or
duplicate reading.) It was found convenient to construct
28 analytical sticks, 2.75 inches long by 0.25 inch wide on
a 2.75 inch by 7 inch sheet of backing.
To adhere the adsorbent at zone (b), one of
the brown paper layers was scored 0.75 inch from one long
edge and the 0.75 inch x 7 inch strip of brown paper removed
to expose the white sticky center.
To prepare the adsorbent for zone (b), 1 part
by wt. of activated carbon powder was mixed with 2 parts
by wt. cellulose powder ~grade for thin layer chromatography),
1 part by wt. plaster of paris and enough water to form a
slurry. This mixture was spread as a 0.5 mm layer on the
back of a glossy medium weight polyethylene-coated photo-
graphic enlarging paper which had been fixed and washed.
- 20 This adsorbent layer was allowed to dry overnight and cut
into 0.125 x 8.5 inch strips. One of these strips was
adhered carbon layer up on the sticky backing next to the
:.
brown paper edge as shown in the drawing at c (enlarging
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i~ paper) and d (carbon layer). In this way, the amount of
; adsorbent applied in zone (b) was approximately 0.4 mg. (In
use when this adsorbent is contacted by about 7 microliters
of liquid extract moving through by capillary action, an -
approximate ratio would be attained of 1 mg carbon per 17.8
microliters of liquid extract, or 0.7 g carbon per 12.5 ml
liquid extract or per g of seed, which previously was found
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to adsorb the inhibitor without appreciably adsorbing the
glucose.)
The remaining area of the exposed sticky
surface was then covered by Whatman No. 1 filter paper as
wick, with a slight (1/32 inch) overlap of the adsorbent to
provide contact for capillary action (see drawing e).
The 2.125 x 7 inches of brown paper remainin~
was pulled off and a strip of glucose test paper ~Tes-Tape
{trademark} Eli Lilly Co.) 3/16 x 7 inches, was applied with
a slight overlap (1/32 inch) of the adsorbent to provide for
capillary action (see drawing f). Next a 1/16 x 7 inch
strip of the same filter paper was applied with a slight
overlap of the downstream edge of the glucose test paper, to
allow capillary action to continue to beyond the glucose test
paper and provide for uniform colour development in the test
paper (see drawing g). Because the blue o-tolidine complex
,~ is water-soluble and moves with the capillary action, the
width of this final wick was limited to about 1/16 inch,
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minimizing elution of the colour complex from the test paper.
This final downstream wick is optional but preferred. Instead
of being normal wick material, this final strip could be blue
litmus paper ~or similar indicator paper) which would turn
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~ pink when contacted by the aqueous extract which is acidic
",!~ (pH about 5 - 5.5). This colour change is useful in showing
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when capillary action is complete, and may also serve to
indicate that the stick has been used for a test in which
negligible colour development occurred.
: -
The remainder of the exposed white backing was
covered with a non-absorbent paper (see drawing h) to provide
for handling and labelling. One protective covering of black
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plastic non-absorbent tape (see drawing at i) was placed over
the adsorbent layer overlapping the glucose test paper
slightly, and extending a short distance (0.25 inch) over the
wick (e). This tape protects the fragile adsorbent layer and
also demarks the level to which this analysis stick is to be
submerged in the aqueous extract. By not submerging this taped
area or above, the aqueous extract is allowed to move by
capillary action up about 0.25 inch of filter paper and through
the adsorbent layer before entering the glucose test paper. A
second covering of black plastic non-absorbent tape 0.25 x 7
inches was placed above the glucose test paper (see drawing
at j) to contrast and demark the colour development zone (c)
and facilitate the colour reading.
Upon completion of this assembly the sheet
was cut into 28 analysis sticks of 0.25 x 2.75 inches. These
sticks were used to determine glucosinolate content after
hydrolysis to glucose, in various oilseeds. A total of
12.5 ml of water was added per g of crushed seed to form the
` aqueous extract and each stick submerged at the wick end up
to the edge of the black tape. Full chemical determinations
were run on the same extracts using the thiourea method of
L.R. Wetter, which involves conversion of isothiocyanates
and oxazolidinethiones to their thiourea derivatives in
ammonia ethanol. The W absorption of these derivatives was
measured at 245 nm and converted to mg equivalents of
3-butenyl isothiocyanate per ~m of oil-free meal. Results
are summarized in the following Table.
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Test-Stick rating Gluco-
sinolate
Avg~ Range conten~
Brassica napus
Tanka 6.2 5-7 17-3
Midas 5.8 5-7 17.2
Golden 6 . o 5-7 lG . o
Turret 5-8 5-7 ~5-4
Zephyr 5~Q 4-6 1)~
Target 6.2 5-7 1~.2
Nugget 5-6 5-7 14.1
Oro 4.8 4-5 14.0
Tower 1.2 1-2 2.4
S7N71-1788 1.0 1 1.6
Bronowski 1.0 1 1~3
Brassica campestris
R-500 6.6 6-7 14.7
Echo 3-6 ~002
Polar 5.0 4-6 10.1
Span 4.8 4-6 9.
Torch 5- 5 9-~
Arlo 4.6 4-5 8.6
CZY3-1820 1.0 1 1.
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Brassica juncea
.
Stoke 6.2 5-7 16.6
Lethbridge 22A 5.2 4-6 14.
Ekla 6.8 6-7 1l~.~
Commercial Brown 6.0 5-7 12.V
5% Midas in Tower* 1.4 1-2 ~-3
10% hlidas in Tower 1.6 1-3 3-3
15% Midas in Tower 2.2 2-3 3.7
20% rlidas in Tower 3.4 3-4 4.3
40% Midas in Tower 4.2 3-5 7-3
+Average of 5 determinations rated on the 0-9 rating scale, O repre-
senting no color change, 2, 4, 6 and 8 corresponding to the color
standards on the Tes-Tape pack~ge.
~Avera~e of 2 determinations by the thiourea method expressed as mg
equivalents of 3-butenyl isothiocyanate per gram of oil-free meal.
*Admixture by weight.
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The test stick method was consistently able to identify
low glucosinolate cultivars. Tests with admixtures of seeds
of high and low glucosinolate content confirmed that the
test stick method could consistently detect contamination
of low glucosinolate seed by high glucosinolate seed when
the overall glucosinolate was raised to 3 mg or more
expressed as equivalents of 3-butenyl isothiocyanate per g
of oil-free meal.
The test apparatus is thus very useful for
identifying seed of low glucosinolate cultivars throughout
the transportation and marketing system. It is anticipated
that the test apparatus will be used by farmers to verify
that the seed they are sowing is low in glucosinolate
content, by truckers to verify that the seed they are hauling
is low in glucosinolate content, by crushing plants and
country elevators to identify deliveries of low glucosinolate
seed, and generally throughout the processing and marketing
system to segregate seed of high and low glucosinolate content.
Thus many tests will be done under field conditions and it
would be desirable to have a kit including the test apparatus
to facilitate the entire assay.
A kit has been designed which cPntainS
; disposable plastic containers able to withstand crushing of
the seed contained therein and within which water can be
admixed with the crushed seed. Separate measuring containers
or other means for measuring the seed sample and water are
also provided. Optionally, the kit can include a sample o
known low-glucosinolate content seed which can serve as a
standard for comparison, and also as a source of myrosinase
where myrosinase-free non-hydrolyzed glucosinolate compositions
are to be assayed. For instance, for analysis of commercial
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rapeseed meal a source of myrosinase must be added since the
seed-borne enzyme activity is destroyed by commerical oil
extraction processing. Alternatively, myrosinase itself or
another source thereof (such as an oil-free non-heated low-
glucosinolate meal) can be present. For convenience, in kits
intended for use with commercial meals or other non-hydro-
lyzed, myrosinase-free samples only, the source of myrosinase
could be placed in the plastic container to be used for
crushing, kneading, mixing, etc. The kit may also contain
seed crushing means able to exert sufficient compression or
impact to crush the seed (e.g. hammer or screw compression
means). The test apparatus, test procedure, and colour
standards for the glucose test tape would always be included.
With such a kit the assay would be performed as follows.
A small graduated container would be filled to
the mark with seed (e.g. 1 g) and the seed transferred to a
sturdy plastic bag (suitably having a zipper-type closure).
The seed in the closed bag would be crushed, e.g. with
hammer impacts, taking care not to rupture the bag. A
measured amount of water (e.g. ~.5 ml) would be transferred
to the bag and the crushed seed and water kneaded to form a
paste, kneading or mixing continuing for about 2 min. to
allow for enzyme action on the glucosinolates. A further
measured amount of water (e.g. 10 ml) would then be added to
the bag and the contents mixed to form an extract. Only the
wick portion (zone (a) ) of the test apparatus would be
contacted up to the mark thereon. When the extract has moved
up through the wick, adsorbent and glucose test paper, the
test apparatus is separated from the extract and let stand
for about 3 min. Green colour development in the yellow
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glucose test paper may be rated by comparison to colour
produced by seed samples of known glucosinolate content
or by comparison with the colour standards provided. On
a rating scale of 0 to 9 where 0 represents no colour change
and 2, 4, 6 and 8 correspond to the colour standards provided,
low glucosinolate seed should rate no higher than 3. The
plastic bag and ana~ysis test apparatus are discarded after
use.
As described in the Example above, one very
convenient and compact analysis stick would have the approxi-
mate dimensions measured consecutively in the downstream
. direction starting from one end:
:: sample-receiving zone (a) 12.7 mm
wick length between zones (a) and (b) 3.2 mm
i total wick length 15.9 mm
~ adsorbent zone (b) 3.2 mm
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test zone (c) 4.8 mm
with the width being approximately 6.4 mm and the weight of
carbon adsorbent being about 0.4 mg. This size permits use
. ~ 20 of the standard width of the Tes-Tape (3/16 inch or about ~
.~.
!~' 4.8 mm) for the extent of zone (c).
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