4. DESCRIPTION
Field of the Invention:
The present invention relates to preparation of strip for detection of
adulterant in milk. Specifically in present invention, a test strip for detection
of added urea in milk has been developed and method for its use in
detecting added urea in milk is described.
Background of the invention:
Urea is present in blood, urine and milk. Its level in blood reflects
functioning of kidney and high level in blood is toxic. Therefore urea level
in bloodlurine is regularly monitored in patients. A number of methods viz.,
p-dimethyl aminobenzaldehyde method, diacetyl monoxime method,
measurement of change in pH or measurement of carbon dioxide partial
pressure, coupled reaction using urease and glutamate dehydrogenase
enzymes, Hantzsch reaction (European Patent Application No. 801 07452.7),
ion selective electrode, ammonia selective electrode and infrared
spectroscopy (European Patent, 93908832.4) are available for detection and
estimation of urea in blood/urine/milk employing reaction condition in
liquid phase where urea or degraded products of urea reacts with reagents or
brings about change in pH or partial pressure of ammonia or carbon dioxide.
In contrast to plasma or serum or urine, milk is turbid. Methods of urea
estimation extended to milk usually require prior precipitation of milk
protein. These methods usually take more than 60 min and require
expensive equipment and person trained in technical skills. In order to
overcome these limitations, strip based methods for numerous analytes have
been developed. Strip based method for detection of urea in milk (Kumar,
H.; Kumar, A.; Kumari, P.; Jyotirmai, S. and Tulsani, N.B., 2000, A rapid
estimation of urea in adulterated milk using dry reagent strip, Indian
2 9 NOV 208
Journal of Chemical Technology, 7 146-147) and serum (Kumar, H.;
Kumar, A.; Kumari, P.; Jyotirmai, S. and Tulsani, N.B., 2000, A test strip
for the estimation of urea in serum, Indian Journal of Clinical Biocliemistry,
15 124-127) is available and the method requires use of mercaptoethanol
and glutaraldehyde. Mercaptoethanol gives foul smell and glutaraldehyde is
toxic. Further, in this type of strip, there is no uniform colour development
as the regents present on the strip and urea in milk are carried away with the
flow of milk and thus there is more colour intensity at front-line. This
severely affects the readability of the strip and thus decision. Also, an
Indian patent (No. 250500) has been filed for detection of urea in milk. This
is not truly strip based method where reactants are mixed over filter paper.
This test does not use enzymes and therefore can not provide higher order of
specificity.
Object of the Invention:
An object of this invention is to prepare a strip for detection of
adulterated milk with urea.
Another object of this invention is to prepare strip for detection of
urea, based on specific reaction of reagents with urea.
Yet another object of this invention is to provide a method for rapid
detection of 'extraneously added urea' in milk.
Further object of this invention is to prepare a strip which on
immersion in milk results in color change on its reaction with urea in
milk.
Still further object of this invention is to provide a strip for detection
of adulteration of milk with 'urea', which does not involve any
instrument.
Yet another object of this invention is to prepare a strip for detection
of adulteration of milk with 'urea', using non-toxic reagents.
Still another object of this invention is to provide a strip for detection
of adulteration of milk with 'urea' which enables on the spot check of
urea adulteration in milk at home by consumers or under field
condition or milk collection center.
Summary of the Invention:
To achieve the aforementioned objects, the present invention describes
preparation of strip for detection of added urea in milk. The preparation of
strip requires following materialslreagents.
a) Support rnatrix
b) Enzyme
c) Chrornogen
d) Flow resisting agent
This invention further describes a method for the detection of added urea in
milk using said strip. The developed method uses the art, wherein colour is
uniform and thus decision making is better.
Brief description of the accompanying drawings:
Other features as well as the advantages of the invention will be clear from
the following description.
In the appended drawings:
Figure 1. Various steps in the detection of added urea in milk using strip
Figure 2. Change in colour of strip with increasing amount of urea in milk
Detailed description of the invention:
The following description is of exemplary embodiments only and is not
intended to limit the scope, applicability or configuration of the invention in
any way. Rather, the following description provides a convenient
illustration for implementing exemplary embodiments of the invention.
2 9 NOV 2013
Various changes to the described embodiments may be made in the function
and arrangement of the elements described without departing from the
scope of the invention.
Preparation of strip requires support matrix which is a chromatographic
paper, an enzyme which is urease from Jackbean (Canvavlia ennsformis) (>
45 unitslmg), a chromogen which is a phenol red sodium salt and a flow
resisting agent which is sodium alginate. The developed method uses the
art, wherein colour is uniform and thus decision making is better. The
analytes do not spread over the strip with the flow of the milk and thus
colour intensity is uniform throughout the strip and thus helps in better
decision making.
Procedure for preparation of strip
The invention provides a procedure for preparation of strip for detection of
'urea' in milk, comprising following steps:
a) Preparation of phosphate buffer of 100 mM and pH 6.5, by dissolving
1.039 g of sodium dihydrogen orthophosphate and 0.473 g of disodium
monohydrogen orthophosphate in 100 ml of distilled water.
b) Preparation of 'enzyme solution' by mixing 10 mg of urease (from
Jackbean, >45 unitslmg) and 250 mg of sodium alginate in a 10 mI of
phosphate buffer till a uniform honey like consistency is reached.
c) Preparation of chromogen solution by dissolving 100 mg of phenol red
sodium salt in 100 ml of distilled water.
d) Soaking chromatographic paper in an 'enzyme solution' for 5 minute or
more duration, followed by drying it at room temperature (2530°C) for
2 hours.
e) ARer complete drying, enzyme containing chromatographic containing
enzyme soaked in chrornogen solution for 5 minute or more, followed
by drying it at room temperature (25-30°C) for 2 hours.
f) The treated chromatographic paper is yellow in colour and the same can
be cut into strips. The strips can be stored in a plastic bag at room
temperature or under refrigerated condition.
Procedure for detection of 'extraneously added urea' in milk using
paper strip
The procedure for detection of adulterated milk with urea using paper strip
is very simple and it just requires momentary dipping of strip in milk.
immediately afterwards, the strip is placed on dry non-absorbent surface.
The colour of strip is noted.
Principle:
When milk and thus urea (present in milk) comes in contact with the paper
strip, the urease present in the strip hydrolysed the urea into ammonia and
carbon dioxide, the produced ammonia being alkaline in nature raises the
pH of strip. The increase in pH is recognized by colour change of the strip
from yellow to redlpink by chromogen which is phenol red. Therefore, the
colour of the strip will change from yellow to redlpink depending on
amount of urea present in milk. The strip further comprises flow resisting
agent which is sodium alginate in the present invention. Sodium alginate is
a hydrocolloid which holds the milk on the strip due to its water binding
capacity and thereby retards flow of milk through the strip. This
phenomenon results in certain advantages including higher amount of
available milk and thus urea per unit area of strip and formation of sharp &
uniform colour band, etc. Sodium alginate on the strip when being dried,
forms a matrix which entraps urease enzyme uniformly. This also prevents
washing away of urease by holding it on the paper during soaking in
chromogen solution.
Analysis of results:
The results are analysed by comparing colour produced on the strip as a
result of reaction of pure milk vis a vis adulterated milk with detecting
reagents present on the strip and the resulting colour on the strip indicates
the presence or absence of extraneously added urea in the milk sample.
The paper strip specifically detects colour change in milk samples having
900 ppm of the urea.
The invention will now be explained with the help of following examples.
However, the scope of the invention should not be limited to these examples
as the person skilled in the art can easily vary the proportion of the
ingredients and combinations.
Example 1:
Qualitative Test:
A piece of strip is dipped into a milk sample up to a certain depth for few
seconds and kept aside on a dry non-absorbent surface. A change in the
colour of the strip is observed after 120 seconds. Appearance of peach
colour indicates the absence of extraneously added urea. Whereas, redlpink
colour of the strip indicates presence of an extraneously added urea in milk.
Figure 1 and Figure 2 explain the use of strip for detection of urea in milk
and development of colour in the strip.

2 9 NOV 2013
We claim
I. A strip for detection of 'urea' comprising of support matrix coated with
an enzyme, chromogen and flow resisting agent and the method for
detection of urea in milk.
2. Enzyme as claimed in claim 1 is urease.
3. Chromogen as claimed in claim 1 is phenol red or its sodium salt.
4. Flow resisting agent as claimed in claim 1 is sodium alginate.
5. Support matrix as claimed in claim I is chromatographic paper.
6. A strip for detection of urea as claimed in claim I is prepared by first
soaking chromatographic paper in enzyme solution prepared by
dissolving 10 mg urease (> 45 unitslmg), 250 mg sodium alginate in 10
ml phosphate buffer (100 mM, pH 6.5) or any other buffer of same or
similar pH and then soaking in chroinogen solution comprising of 100
mg phenol red - sodium salt in 100 ml distilled water.
7. A method for detecting urea in milk as claimed in claim 1 using strip as
claimed in claim 1 to claim 6 wherein prepared strip is dipped in test
solution, then placed on non-absorbent surface followed by matching of
colour as shown in Figure 2.
Dated 2~44Iayo f 6' d- 20 13
J
Director,
National Dairy Research Institute
(Indian Council of Agricultural Research)
Krishi Bhawan. New Delhi-110001, India