Journal of Dairy Research Validation of strip tests for the rapid screening
cambridge.org/dar
Research Article
Cite this article: Fagnani R, de Lira FM and
Rosolem CP (2019). Validation of strip tests for
the rapid screening of ethanol residues in milk.
Journal of Dairy Research 86, 464–466. https://
doi.org/10.1017/S0022029919000827
Received: 19 June 2019
Revised: 18 September 2019
Accepted: 18 September 2019
First published online: 14 November 2019
Keywords:
Adulteration; alcohol; fraud; detection
Author for correspondence:
Rafael Fagnani, Email: rafaelfagnani@hotmail.
com
© Hannah Dairy Research Foundation 2019
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of ethanol residues in milk
Rafael Fagnani, Fernanda Montanholi de Lira and Carla Prado Rosolem
Science and Technology of Milk and Dairy Products Master’s Degree. Unopar University, Marselha Street 591,
Londrina, PR, 86041-140, Brazil
Abstract
The experiments reported in this Research Communication aimed to determine if a domestic
strip test to detect ethanol residues in breast milk (Milkscreen®) is comparable to a previously
established official method for detecting ethanol residues in milk. The two methods are exam-
ined in terms of overall sensitivity, robustness against storage and acidity and selectivity
against formaldehyde residues. Here, Milkscreen® provided advantages, with faster results
(2 min), good sensitivity (≥0.017%), no false results due formaldehyde residues and equal
robustness against storage, but with lower sensitivity in acid milk samples. In summary,
strip tests for the rapid detection of ethanol residues in breast milk can be used for screening
purposes by dairy manufacturers, combining it with the official method to make a final
diagnosis.
Milk tampering is a major issue in the global dairy industry. One of the most frequent adul-
terations is the addition of extraneous water, which can be accompanied by various ways to
disguise it, such as addition of melamine, urea, sodium chloride, urine, sucrose, starch and/
or ethanol (Wanjala et al., 2018). It is estimated that the addition of ethanol occurs at a fre-
quency of 7.7% (Mareze et al., 2015). The official method for detecting ethanol residues in
milk is labor-intensive, requiring specific glassware and specific reagents. The estimated aver-
age time of this analysis is 15 min per sample. As a result, a rapid ethanol detection method is
highly demanded, and it could be useful for official control entities and for quality control in
dairy manufacturing (Brasil, 2018a, 2018b).
To detect the presence of alcohol in human milk, some countries already provide domes-
tic testing. In the US there are strip tests for detecting alcohol in breast milk, sold in phar-
macies under the name MilkScreen®. This test promotes better quality of life for mothers,
allowing them to occasionally consume alcoholic beverages without endangering the infant’s
health by avoiding consumption of breast milk with alcohol residues. According to the
manufacturer, the test pad will change color if the alcohol is present at, or above,
13.1 mg/dl.
Other tests are also available including Safe Milk™ and the PREGMATE One Step Alcohol
Breast Lek Test™, both with precision of 20 mg/dl. Although manufacturers do not identify
the chemical principle of these tests, they are likely to be based on an enzymatic oxidation
reaction with alcohol dehydrogenase, utilizing several reactions and having as a closing
point a color change after 2 min (Christopher and Zeccardi, 1992).
Considering the sensitivity of these strip tests and the compositional similarity between
milk and breast milk, it is likely that the MilkScreen™ could identify ethanol residues in non-
human milk samples (ie dairy industry milks) and in the future be used as an alternative to the
official test. On this basis, we aimed to determine if the MilkScreen™ is a suitable tool to
detect extraneous ethanol in bovine milk.
Material and methods
To evaluate if Milkscreen® can detect extraneous ethanol mixed in milk, ethanol 92 ml/dl from
Dinâmica (Diadema, Brasil) was diluted in fresh raw bovine milk to provide working samples
with final volume of 100 ml and concentration of 0.00 ml/dl (control), 0.010 ml/dl, 0.017 ml/
dl and 0.020 ml/dl. Each sample was homogenized in vortex for 7 s and after 30 min the ana-
lyses were performed.
For the Milkscreen® test, the strip was plunged in the milk sample for ten seconds, and after
2 min, the reading was performed. With the assistance of a color scale supplied by the manu-
facturer, the reading was performed observing any color change in the strips.
For the official alcohol test 100 ml of the sample was transferred to the Büchner flask, add-
ing 3 ml of 3 ml/dl antifoam solution (Tec-Lab, Brazil). The Büchner flask was boiled for
5 min and the steam was conducted through a latex tube and a Pasteur pipette to a test
tube containing 2 ml of sulphochromic solution. Finally, the yellowish coloration of the
 Journal of Dairy Research 465

Table 1. Absolute frequencies of positive and negative results in milk samples added with ethanol evaluated by Milkscreen® and by official method

Concentration of ethanol

0 ml/dl 0.010 ml/dl 0.017 ml/dl 0.020 ml/dl

Performed 30 min after ethanol addition

Official method (−/+) 10/0a 0/10a 0/20a 0/10a
a b a
Milkscreen® (−/+) 10/0 10/0 3/17 0/10a
Performed 48 h after ethanol addition
Official test (−/+) 10/0a 2/8a 0/10a –
Milkscreen® (−/+) 10/0 a
10/0 b
3/7a –
Performed in acid milk samples
Official test (−/+) 13/0a 13/0a 2/11a –
Milkscreen® (−/+) 13/0 a
13/0 a
13/0b –
Concentration of formaldehyde
0 ml/dl 0.005 ml/dl 0.0075 ml/dl 0.010 ml/dl
Performed in milk samples with formaldehyde
Official test (−/+) 3/0 3/0 0/3 0/3
Milkscreen® (−/+) 3/0 3/0 3/0 3/0
Proportions followed by distinct letters differed in test χ2 (P < 0.05); (−/+) absence/presence of ethanol detection.

sulphochromic solution represented the negative result for the
presence of ethyl alcohol, while the appearance of the green
color was considered positive (Brasil, 2018a).
The analyses were repeated 10 times for each alcohol concen-
tration, except for the lowest detection concentration reported by
the Milkscreen® manufacturer, which was repeated 20 times. At all
concentrations, the proportion of positive and negative results
from each test was compared using the χ2 test with Yates correc-
tion and P ≤ 0.05.
In order to evaluate the robustness of the methods against
storage of milk during 48 h under refrigeration, the same con-
centrations used for the sensitivity evaluation were stored in a
volumetric flask for 48 h at 2 °C to 8 °C prior to the tests.
After the mentioned period, the tests were performed as
reported above.
In order to evaluate the robustness of the methods against
the acidity, the same samples used for the sensitivity assess-
ment were stored at 28 °C until the titratable acidity naturally
reached 0.20 g/dl of lactic acid concentration. Then, the ethanol
detection was assessed by Milkscreen® and by official method
as reported above.
The robustness tests were repeated in triplicate. In case of
inconsistency of results, a new test was performed with another
ten repetitions to make it possible to compare the proportion of
positive and negative results by the χ2 test with Yates correction
and P ≤ 0.05.
In order to evaluate the selectivity of the methods against for-
maldehyde residues, different concentrations of formaldehyde
38 ml/dl (Dinâmica, Brazil) were diluted into a total volume of
100 ml of raw milk. The final concentrations of formaldehyde
used were 0.005, 0.0075, 0.010 ml/dl. After 30 min the strips
were immersed and read as described previously. The selectivity
tests were repeated in triplicate. In case of inconsistency of results,
a new test was performed with another other ten repetitions to
make it possible to compare the proportion of positive and nega-
tive results by the χ2 test with Yates correction and P ≤ 0.05.
Results and discussion
Milkscreen® was able to detect ethanol residues from concentra-
tions of 0.017 ml/dl. On the other hand, the official test was
more sensitive, being able to detect ethanol residues from concen-
trations of 0.010 ml/dl as described in Table 1. Considering the
results of the effect of ethanol on the milk’s freezing point, con-
centrations of 0.010 and 0.017 ml/dl ethanol respectively allow
the fraudulent addition of water in concentrations of 0.75 and
1.29 ml/dl, keeping the freezing point parameters within specified
limits for an authentic milk.
However, the amounts of water added by fraudsters are usually
higher. In 2007 researchers detected fraud by the addition 3.6 ml/
dl of water in raw milk (Barbosa et al., 2007). In this case, it would
be necessary to add 0.05 ml/dl ethanol to the milk to keep the
freezing point parameters within specified limits for an authentic
milk and to disguise the dilution of the milk. In 2013, a Brazilian
investigation called ‘Compensated Milk Operation’ and conducted
by the Federal Prosecution Service discovered that milk producers
acted fraudulently by the addition of 10 ml/dl water (Globo,
2013). To disguise this adulteration, it would be necessary to
add about 0.15 ml/dl ethanol. Based on this, the Milkscreen®
could be performed on the milk reception laboratories along
with the screening tests used in dairy products.
Even after storage of the milk samples for 48 h under refriger-
ation, the sensitivity of both tests remained unaltered, remaining
0.010 ml/dl for the official test and 0.017 ml/dl for the
Milkscreen® (Table 1). Thus, it is possible to detect ethanol resi-
dues even after 48 h of milk storage, conditions allowed by current
Brazilian regulations (Brasil, 2018b).

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 466
Regarding the detection of ethanol in acid milk samples,
both tests had variations in sensitivity. The official test failed
to detect ethanol residues in concentrations of 0.010 ml/dl
and began detecting them in concentrations of 0.017 ml/dl.
In its turn, the sensitivity of the Milkscreen® also decreased
with milk acidification, being unable to detect ethanol residues
even in concentrations of 0.017 ml/dl (Table 1). In the samples
with 0.010 ml/dl of ethanol, it would be possible to add
0.076 ml/dl water. In its turn, the ethanol added at the concen-
tration of 0.017 ml/dl would allow addition of up to 1.29%
water. Notwithstanding the decrease in sensitivity of both
tests, the acidity could be detected during commercial milk col-
lection through the test of alcoholic stability and/or in the
reception of the dairy through the titration of acidity, manda-
tory tests before the industrial processing of the milk (Brasil,
2018b).
Regarding the selectivity of the tests due to formaldehyde resi-
dues, the Milkscreen® was more robust compared to the official
test, without changing the sensitivity results in concentrations
up to 0.010 ml/dl of formaldehyde. In its turn, false-positive
results in the official test were observed from 0.0075 ml/dl formal-
dehyde, as can be seen in Table 1. The official alcohol test is not
specific for alcohol detection, as the reduction of Cr+6 to Cr+3
occurs in the presence of aldehyde groups, which, in addition
to being formed in the oxidation of primary and/or secondary
alcohols, may also be from the formaldehyde molecule (Tojo
and Fernández, 2006).
In conclusion, with good sensitivity and robustness,
Milkscreen® is comparable to the official method to detect extra-
neous ethanol in fresh raw milk, working even in sour milk and
without false-positive results due to formaldehyde residues.
These results support the validity of Milkscreen® strips for
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Rafael Fagnani et al.
screening purposes by dairy manufacturers, combining it with
the official method to make a final diagnosis.
Acknowledgements. The authors acknowledge the Fundação Nacional de
Desenvolvimento do Ensino Superior Particular (FUNADESP) for financial
support.
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