ARTICLE IN PRESS

Lebensm.-Wiss. u.-Technol. 36 (2003) 567–571

Dry-reagent strips for testing milk pasteurization

Sandeep K. Sharmaa, Neeta Sehgalb, Ashok Kumara,*
a
Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India
b
Department of Zoology, University of Delhi, Delhi 110 007, India
Received 30 July 2002; accepted 18 February 2003

Abstract

Biostrips have become more popular for diagnosis of various diseases as well as for testing of different chemical and biochemical
parameters. Alkaline phosphatase (ALP) is an enzyme naturally present in raw milk, which is used as an indicator for proper milk
pasteurization. Nonpasteurized or raw milk contains ALP, which causes intra-abdominal bacterial infection after drinking the milk,
whereas after pasteurization, ALP is denatured. Therefore, milk industries test the milk after pasteurization using conventional
methods such as colorimetric and fluorescence. Our attention was drawn to develop quick, simple and economical test using dry-
reagent strips for detection of ALP activity in milk. It is based on ALP reaction with p-nitrophenyl phosphate in the presence of
water to liberate p-nitrophenol and inorganic phosphate. p-Nitrophenol on reacting with a specific chromogen changes the colour of
the strip from light blue to green, which is visualized by the naked eyes. The strip has a sensitivity of >0.5 units/L. The strips may be
used in dairy industries and remote areas where expensive instruments are not available. The strip is stable for more than a year at
room temperature.
r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Alkaline phosphatase; Biostrip; Chromogen; Pasteurization

1. Introduction Various milk industries test the milk after pasteuriza-

tion using conventional chemical methods (Angelino,
Milk is one of the essential nutrients for human and Christen, Penfield, & Beattie, 1999; Lombardi,
animal consumption. Alkaline phosphatase (ALP) Avallone, D’angelo, Mor, & Bogin, 2000; Scintu, Daga,
(Garen & Levinthal, 1960; Gettins, Metzler, & Coleman, & Ledda, 2000; Vega-Warner, Gandhi, Smith, &
1985), g-glutamyl transferase (Anjos, Machado, Ferro, Ustunol, 2000) for detection of ALP. The Fluorophos
Otto, & Bogin, 1998), lactoperoxidase (Mortier, Breck- ALP test system (Advanced Instruments, Inc) and the
man, Cartuyvels, Renterghem, & Block, 2000; Marks, Charm ALP (Charm Sciences, Inc) are relatively more
Grandison, & Lewis, 2001) and leucine arylamidase sensitive new instrumental procedures for detecting ALP
(Gerard, Bernard, Asia, & Didier, 2002) are native activity in milk (Angelino et al., 1999). An inert
enzymes of milk. These enzymes have thermal resistance substrate containing a phosphate group is converted to
greater than that of the most heat-resistant nonspore- either a fluorescent or light-generating compound as
forming pathogens commonly found in milk. ALP is read by the instrument is proportional to the activity of
slightly less heat labile (71.6 C for 15 s) than most ALP in the sample (Rocco, 1990; Black, 1992). The level
pathogenic bacteria, thus loss of ALP activity confirms of ALP in milk is always found variable depending on
proper pasteurization of skimmed or whole milk. the source of raw milk (6.0–28 units/L). Whereas,
Therefore, ALP is used as an indicator of proper milk the pasteurized milk contains ALP activity of about
pasteurization. Nonpasteurized milk has the presence of 0.001–0.006 units/L. Higher ALP activity may indicate
ALP, which could cause intra-abdominal bacterial serious deficiencies in the pasteurization process.
infection and intestinal malfunction after consuming. Recently, a rapid quantitative reflectometer RQ flex
ALP gets denatured in the process of pasteurization. plus is developed by Merck Inc. to test pasteurization of
milk using ALP test strips and test kit (Merck, 2002).
*Corresponding author. Fax: +91-11-27667471. The reflectometer RQ flex system is quite expensive, as it
E-mail address: ashokcbt@rediffmail.com (A. Kumar). requires instrument as well as disposable test strips for

0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0023-6438(03)00061-6
 ARTICLE IN PRESS
568 S.K. Sharma et al. / Lebensm.-Wiss. u.-Technol. 36 (2003) 567–571

each test. The procedure for testing milk pasteurization 2.4. Preparation of chromogen and pNPP solution
also requires specific temperature (37 C), different
(a) Bromocresol green 0.1 mg/mL was prepared in 1 M
reagents and takes more than 20 min for reading the
Tris-HCl buffer, pH 8.0.
results. Our system does not require any sophisticated
(b) pNPP 0.4 mg/mL was prepared in 1 M Tris-HCl
instrument and is based on development of colour on
buffer pH 8.0.
strips, which is visualized by naked eyes. The change in
colour depends upon the concentration of ALP activity Solutions (a) and (b) were mixed in 1:1 ratio for
present in milk samples. The response time of the strips coating the matrix Whatman no. 1 filter paper.
is 2 min and cost of 1000 tests will be only approximately
US$2.
2.5. Preparation of dry-reagent test strip
All reported methods are expensive, cumbersome and
require many chemicals to detect ALP in pasteurized
The mixture of chromogen and pNPP solution was
milk. We have developed different types of dry-reagent
immobilized on Whatman no. 1 filter paper (4  30 cm2)
strips in our laboratory for the detection of various
and dried at 30 C for 1 h in a humidity-free chamber
chemicals and biomolecules (Kumar, Tulsani, & Singh,
(Guilbault, 1984; Guilbault & Neto, 1985; Barker,
1997; Kumar & Tulsani, 1997; Kumar, Kumar, Tulsani,
1987; Kumar et al., 2000). After complete drying, the
& Joshi, 1998; Kumar, Kumar, Kumari, & Tulsani, 2000;
colour of the Whatman paper became light blue.
Sharma, Bala, Tulsani, Sehgal, & Kumar, 2002a, b;
The paper was cut into several pieces of 5 mm width
Tulsani et al., 2001). Here, we describe a quick, simple
using specially designed strip cutter. The plastic sheet
and economical dry-reagent strip technique for detection
was cut into the size of 9.0  90 cm2. The strips of
of ALP in pasteurized milk samples.
the reagent-coated matrix were pasted along the edge
of the plastic sheet using Vamicol (a nonreactive
adhesive), diluted 1:10 with distilled water. The sheet
2. Materials and methods was then dried in a humidity-free chamber for 2 h. After
complete drying, the sheet was cut into 0.5  9.0 cm2
2.1. Materials pieces in such a manner that one end of the strip
has a reagent pad and the other end is free for handling.
Tris(hydroxymethyl)aminomethane, bromocresol These strips were stored in brown bottles containing
green and p-nitrophenyl phosphate (pNPP) were obtained silica gel bags as desiccant at 4 C and at room
from Sigma Chemicals, USA. ALP (Escherichia coli) temperature (30 C).
was from Pharmacia LKB Biotechnology, USA. Ad-
hesive Vamicol was from Vam Organic Chemicals Ltd.,
New Delhi, India. Matrices were purchased from
Whatman Co., UK. Plastic sheets were obtained from 3. Results and discussion
local market. All solutions were prepared in triple
distilled water. There are various analytical methods reported for
detection of ALP in pasteurized milk. Dry-reagent strip
method is developed in our laboratory. In this strip,
2.2. Assay of ALP immobilized chromogen and substrate pNPP on react-
ing with ALP in milk sample changes the colour from
The enzyme activity of ALP was estimated according light blue to green. Milk from all mammals contains
to the method of Garen and Levinthal (1960). The ALP, although the amount varies from species to
enzyme ALP for standardization was taken from E. coli species (Fernley, 1971; Linden, Mazeron, Michalowski,
as it is more heat stable than other sources. One unit of & Alais, 1974; Williams, Nottingham, Petroff, &
ALP hydrolyses one micromole of pNPP per minute at Veldhoven, 1991). Most of the pasteurized milk supplied
25 C, pH 8.0, under the specified conditions. by the milk industries, is mixed and comes from buffalo,
cow, goat, etc. The nonpasteurized raw milk (ALP
2.3. Estimation of ALP activity in milk samples activity >0.5 units/L) changes the colour of the strip
from light blue to green, whereas pasteurized milk (ALP
The fresh milk samples from different mammals such activity o0.5 units/L) does not change the colour of the
as buffalo, cow and goat were collected in clean strip (Fig. 1). The response time of the strip is 2 min and
containers and centrifuged at 2000 rpm for 5 min at the strip test gives a ‘‘Pass or Fail’’ result. If ALP
10 C. The top lipid layer was removed by pipette and activity is >0.5 units/L (green colour of strip) the
ALP activity was estimated in pasteurized as well as sample is failed and if it is o0.5 units/L (no change in
in nonpasteurized raw milk samples according to the the light blue colour of strip) the sample is passed. It
method of Garen and Levinthal (1960). does not give exact quantitative result.
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Different types of matrices such as Whatman filter useful for qualitative detection of ALP in milk. Different
paper, nylon paper and ordinary papers have been milk samples collected from the local market were tested
tested. Whatman paper no. 1 is more suitable for with these strips and the results are shown in Table 1.
immobilization of chromogen and substrate. The strips The raw milk samples collected from local sellers show
are stable at room temperature for several months if the colour (i.e. nonpasteurized), whereas industrial milk
stored under humidity-free conditions. The strip is samples do not show any change in the colour of the
strip suggesting that the milk is properly pasteurized.
When pasteurized milk is adulterated with raw milk,
Alkaline phosphatase the ALP activity also increases proportionately. The
Light Blue Green
colour of the strip changes depending upon the
Original strip colour Non-pasteurized sample
concentration of raw milk added to the pasteurized
(Raw milk) milk as shown in Table 2. Similar results are reported
with Reflectoquant (RQ flex plus) as well as with EU
Fig. 1. Colour chart for ALP strip. The pasteurized milk (ALP activity
o0.5 units/L) does not change the colour of the original strip, whereas
reference method. Fluorophos ALP test system also
nonpasteurized raw milk (ALP activity >0.5 units/L) changes the reported same results (Rocco, 1990). The level of ALP in
colour of the strip from light blue to green. raw milk may vary depending on the source of raw milk.
The values in the table are in good correlation with RQ
flex system (o1.0 units/L ALP in pasteurized sample).
Table 1 The colour of dry-reagent test strip changes on
Detection of pasteurization of milk available from different sources by
adulteration of 1% raw milk in pasteurized sample
strip
(>0.56 units/L ALP in pasteurized sample).
Milk sample ALP activity Pasteurization test The sensitivity of the strip is above 0.5 unit/L of ALP
(units/L)a (strip colour)b
activity. The sensitivity of the strip is tested with
Britannia Milk Manc 0.002–0.004 Light blue ( ve) different concentrations of ALP prepared in completely
Mother Dairyc 0.001–0.005 Light blue ( ve) pasteurized (no detectable ALP activity) milk (Table 3).
Delhi Milk schemec 0.001–0.004 Light blue ( ve) If ALP activity is >0.5 unit/L, the colour of the strip
Parasc 0.003–0.006 Light blue ( ve)
Gopaljeec 0.001–0.004 Light blue ( ve) changes from light blue to green after dipping in the
Local milk 6.00–18.00 Green (+ve) milk samples.
Cow milk 6.00–25.00 Green (+ve) The stability of the strips were monitored alternate
Buffalo milk 6.30–28.00 Green (+ve) month by testing the strips with standard ALP (E. coli)
Goat milk 14.00–20.00 Green (+ve) solution of different serial (6–0.02 units/L) dilutions.
a
ALP activity was also estimated in milk samples according to the The strip was found more stable when stored in brown
method of Garen and Levinthal (1960). The values of ALP were found bottle in the presence of desiccant at 4 C as compared to
variable in different samples. room temperature. The total loss of stability in 1 year
b
The change in dry-reagent strips colour after dipping in milk
samples were recorded after 2 min.
was found to be only 10–20% (Fig. 2). Therefore, the
c
Trade name of milk industries. These industries supply mixed milk strip can be stored in humidity-free conditions at room
after pasteurization. temperature for daily use. All reported methods are

Table 2
Effect of addition of raw milk to pasteurized milk and comparison with other reported methods

% raw milk added Reflectoquant ALP test ALP test with EU reference ALP activity (units/L)c Strips colourd
(units/L)a method (mg/L)b

0.00 Low ( ve) 0.1 ( ve) 0.00 Light blue

0.10 Low ( ve) 1.7 ( ve) 0.00 Light blue
0.15 1.1 ( ve) 3.2 ( ve) — —
0.20 1.5 (+ve) 4.2 (+ve) 0.11 Light blue
0.50 3.5 (+ve) 11.1 (+ve) 0.16 Blue
1.00 7.9 (+ve) 21.8 (+ve) 0.56 Bluish green
10.00 — — 4.50 Light green
20.00 — — 6.70 Green
50.00 — — 10.90 Green
a
Reflectoquant alkaline phosphatase test in milk (Merck, 2002).
b
The regulation of European Union Commission (91/180/EC) states that pasteurized milk must contain ALP activity below 4 mg/mL (Merck,
2002).
c
ALP activity was estimated according to the method of Garen and Levinthal (1960). ALP activity (28 units/L) was added to the pasteurized
sample. The level of ALP activity in raw milk was always found variable. It depends upon the source of raw milk added to the pasteurized milk.
d
The response time for development of colour was 2 min.
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Table 3 ment. This research was supported by the Council of

Sensitivity test of the strip with different concentrations of ALP Scientific and Industrial Research (CSIR), New Delhi,
ALP activity (units/L) Strips colour India.
0.02 Light blue
0.05 Light blue
0.10 Blue
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