Food Control 115 (2020) 107299

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

A novel approach to detect milk adulteration based on the determination of T

protein content by smartphone-based digital image colorimetry
Anna Flavia S. Silva, Fábio R.P. Rocha∗
Center for Nuclear Energy in Agriculture, University of São Paulo, Av. Centenário, 303, 13416-000, Piracicaba, SP, Brazil

ARTICLE INFO ABSTRACT

Keywords: A novel alternative for the detection of milk adulteration by dilution based on the determination of the protein
Dairy industry content is proposed. The proposed method involves the precipitation of the proteins by the salting-out effect of
Food fraud copper sulfate and the proportional adsorption of Cu(II) on the precipitate. Subsequently, photometric mea-
Salting-out effect surements of the remaining amount of Cu(II) by smartphone-based digital colorimetry are conducted to de-
Casein
termine the protein content. The proposed procedure is rapid (sample throughput of 32 assays/h), precise
Photometry
(coefficient of variation = 3.0%, n = 20), environmentally friendly, and allows the detection of as low as 1.0%
v/v water in adulterated milk. The main species used to mask the protein content do not interfere, thus per-
mitting the direct detection of adulteration. The procedure was successfully applied for the classification of milk
samples as compliant and non-compliant as well as for the indirect determination of the protein content in milk.
The quantitative results agreed with those obtained by the reference method (near infrared spectroscopy) at a
95% confidence level.

1. Introduction hypochlorite, dichromate, and salicylic acid) (Das et al., 2016;

Milk is a biological emulsion produced by mammals, in which lipids
are dispersed and partially solubilized by their interactions with pro-
teins, mainly casein, in aqueous phase (Eskin & Goff, 2013). It is one of
the most consumed sources of proteins worldwide, owing to its high
nutritional value and relatively low cost (Eskin & Goff, 2013). In view
of these features, milk fraud and adulteration are common, aiming at
achieving economical gains as well as masking the inappropriate con-
ditions of processing, storage, and transportation (Das, Goswami, &
Biswas, 2016; Handford, Campbell, & Elliott, 2016). In addition to di-
minishing the nutritional value of a milk-based product, these practices
can affect the health of consumers by causing allergies, nausea, diar-
rhea, and other gastrointestinal disorders; respiratory irritation;
bleeding; kidney disorders; and, in more severe cases, cancer and death
(Handford et al., 2016).
Milk adulteration typically involves dilution with, for instance,
water or whey and addition of nitrogen sources (e.g., ammonium salts,
melamine, urea, and non-dairy proteins) to mask the diminution of the
protein content caused by the fraud (Das et al., 2016; Poonia et al.,
2017). Moreover, other substances are added to adjust the visual as-
pects and physicochemical properties of diluted milk, such as sucrose,
sodium chloride, surfactants, vegetable oils, and substances aiming to
increase the milk shelf life (e.g., formaldehyde, hydrogen peroxide,
Nascimento, Santos, Pereira-Filho, & Rocha, 2017; Poonia et al., 2017).
A typical approach to detect adulteration is to determine the protein
content in milk, because it decreases proportionally with dilution.
However, a fraud cannot be detected by traditional approaches, such as
the Kjeldahl and Dumas methods, which measure the total content of
nitrogen and are non-selective to proteins (Simonian & Smith, 2006).
Other limitations of these methods are the high consumption of the
reagents and the time demanded, which lead to low sample throughput
and delayed decisions regarding inappropriate milk samples.
The most common methods to evaluate the protein content in milk
are the measurements of the density, cryoscopic point (also known as
freezing point depression), and refractive index of the whey super-
natant after protein precipitation. These approaches are frequently in-
effective because of the possibility of the masking of the effect of di-
lution by the addition of other adulterants, e.g., salts, sucrose, urea, or
maltodextrin (Rezende, Do Carmo, & Esteves, 2015). Moreover, these
methods present poor sensitivity, which hinders the detection of the
adulterations that typically involve dilutions from 1 to 10% v/v
(Rezende et al., 2015). Alternative methods to determine the protein
content in milk are based on UV–visible spectrophotometry (Simonian
& Smith, 2006), including the Lowry (Lowry, Rosebrough, Lewis, &
Randall, 1951), biuret (Gornall, Bardawill, & David, 1949), Bradford
(Bradford, 1976), and Smith (Smith et al., 1985) methods. The most

∗
Corresponding author.
E-mail address: frprocha@cena.usp.br (F.R.P. Rocha).

https://doi.org/10.1016/j.foodcont.2020.107299
Received 22 January 2020; Received in revised form 13 March 2020; Accepted 6 April 2020
Available online 11 April 2020
0956-7135/ © 2020 Elsevier Ltd. All rights reserved.
A.F.S. Silva and F.R.P. Rocha
remarkable difficulties in the application of these methods in routine
analyses are the high reagent consumption and waste generation, time
demanded, complex sample preparation, and occurrence of matrix ef-
fects (Smith et al., 1985). It is also possible to estimate the protein
content in milk from the absorption of UV radiation (measurements at
205 nm and 280 nm) (Simonian & Smith, 2006); however, selectivity is
a critical aspect. Other approaches to evaluate the authenticity of milk
samples involved determination of non-proteic nitrogen in whey after
protein precipitation (DeVries et al., 2017) or association of videos
acquired with a smartphone to pattern recognition tools (Song, Jiang,
Wang, & Vincent, 2020).
Currently, the standard procedure adopted in the dairy industry to
determine the physicochemical composition of milk and its somatic cell
count is near infrared (NIR) spectroscopy. It leads to the possibility to
pay for milk according to its quality, which is evaluated mainly based
on the balance of the total contents of the proteins and fats in the
sample (Guillou, Pelissier, & Grappin, 1986). NIR spectroscopy is gen-
erally a reagentless procedure, with minimal sample preparation and
low susceptibility to the matrix effects (Guillou et al., 1986; Ribadeau-
Dumas & Grappin, 1989). Its drawbacks are related to the need for
standardized instrumentation (often unavailable), chemometric data
treatment, and constant evaluation of the predictive models, which
require skilled analysts and increase the analysis costs. Furthermore,
estimation of the protein content in milk by NIR spectroscopy needs
calibration with reference samples whose protein contents are fre-
quently determined by the Kjeldahl method (Ribadeau-Dumas &
Grappin, 1989).
Digital images have been exploited in food analysis, e.g., for mon-
itoring the color of food products (Yam & Papadakis, 2004) and para-
meters related to the quality, deterioration, adulteration, and identity
of food (Santos & Pereira-Filho, 2013; Zhang & Suslick, 2007). Analy-
tical measurements are based on rapid image acquisition by a scanner,
webcam, or even a smartphone, followed by conversion of the analy-
tical information to RGB (red–green–blue), HSV (matrix, saturation,
value), or other spaces of color (such as CIE Lab, CMYK, and grayscale).
This approach has been exploited for analytical purposes because of its
practicality, agreement with the principles of green chemistry, cost-
effectiveness, wide availability of instrumentation, and possibility to
achieve high sample throughputs (Santos & Pereira-Filho, 2013). These
characteristics make exploitation of digital images attractive for mon-
itoring milk quality parameters and applications include determination
of antibiotic residues (Lu, Kao, Belkin, & Cheng, 2019; Masawat,
Harfield, & Namwong, 2015), water content and caustic soda (Santos,
Wentzell, & Pereira-Filho, 2012), common adulterants (starch, sucrose,
glucose, and urea) (Luther, Frahan, & Lieberman, 2017), anionic sur-
factants (Acevedo, Lima, Nascimento, & Rocha, 2018), and even Lac-
tobacillus in fermented milk (Borin et al., 2007). Indirect measurements
of the light scattering by digital images were also investigated for the
determination of the fat and total protein contents in milk with data
processing by partial least-squares regression (Kucheryavskiy,
Melenteva, & Bogomolov, 2014).
The aim of this study was to develop a novel, rapid, practical, and
cost-effective procedure to identify milk frauds caused by dilution. The
approach is based on the precipitation of milk proteins by salting out
with copper sulfate and the measurement of the remaining copper(II)
concentration in whey after complexation with ethylenediaminete-
traacetic acid (EDTA). Because the intensity of the blue color measured
via smartphone-based colorimetry is inversely proportional to the protein
content in the sample, it can be an indicator of milk adulteration.
2. Materials and methods
2.1. Samples and solutions
Whole-milk samples (pasteurized and ultra-high-temperature pro-
cessed) were acquired from local markets in Piracicaba city (Sao Paulo,
2
Food Control 115 (2020) 107299
Brazil). The raw milk samples were initially analyzed by the NIR re-
ference method in a laboratory certified by the Brazilian Milk Quality
Council (CBQL).
All the solutions were prepared using deionized water
(resistivity > 18.2 MΩ cm) and analytical-grade chemicals from
Merck, Germany, except when mentioned. Saline solutions
(0.30 mol L−1) were prepared from CuSO4·5H2O, Na2SO4,
Fe2(SO4)3·H2O, (NH4)2SO4·H2O, CaSO4·2H2O, or FeSO4·7H2O (Synth,
Brazil). A solution containing 0.30 mol L−1 CuSO4 and 0.30 mol L−1
Na2SO4 was also used. A complexing solution of 0.25 mol L−1 EDTA
was prepared from its disodium salt (Na2C10H14N2O8·2H2O). A
0.1 mol L−1 2,2′-biquinoline 4,4′-dicarboxylic acid (BCA) (Sigma
Aldrich, USA) solution was prepared in 0.1 mol L−1 ammonium
acetate, and a 2.5% m/v ammonia solution was prepared in water. A
1.0% m/v ascorbic acid solution was employed for copper(II) reduction
by the BCA method.
A β-casein suspension was prepared from 3.30 g protein (Sigma
Aldrich, Switzerland) in 100 mL of 2.5 mol L−1 Ca3(PO4)2 to mimic the
conditions observed in bovine milk. Solutions containing 2.36 g L−1
melamine (Sigma Aldrich, USA), 7.8 g L−1 NH4Cl, or 3.36 g L−1 urea
(Synth, Brazil) were used to evaluate the selectivity of the procedure, as
well as whey, obtained by the addition of 800 μL of chymosin (extracted
from Aspergillus niger 75 IMCU, CHR Hansen, Brazil) in 1000 mL of raw
milk. Reference solutions were prepared by the dilution of a milk
sample, whose protein content was determined as 3.6% m/v by the
reference procedure (AFNOR 2006).
2.2. Apparatus
The precipitation of the milk proteins was conducted in 15-mL
Falcon® tubes with conical bottoms, and the supernatant was trans-
ferred to colorless Eppendorf® tubes. Photometric measurements were
performed directly on these tubes with a smartphone, LG K10 Pro
(equipped with a 13-megapixel camera with resolution 4128 × 2096
pixels and lens aperture f/2.2, equipped with Android 7.1). A free ap-
plication Color Grab (Loomatix, version 3.6.1, 2017), available for
Android systems, was used to convert the images to RGB values. A
styrofoam box (14 cm high, 16 cm wide, and 10.5 cm deep) was
adapted to insert the measurement tubes, and a support was used to
keep the smartphone 9-cm away from them. An LED-based lamp
(Osram Superstar, R50 40 6W E14 30°) was placed on the top of the box
to maintain a constant illumination. The photometric measurements
were performed near the center of the Eppendorf® tubes, with a region
of interest of 32 × 32 pixels.
The absorption spectra were acquired by a UV–vis multichannel
spectrophotometer (Ocean Optics, USA) with a 1-cm optical path glass
cuvette. The reference procedure according to NF ISO 21543 (AFNOR,
2006) was implemented using an NIR spectrophotometer (FOSS
Analytics, Denmark).
2.3. Procedure
The sample processing was based on protein precipitation by the
salting-out effect. An aliquot of 5.0 mL milk was mixed with 1.25 mL of
0.30 mol L−1 CuSO4, following which the mixture was manually
stirred. Other sulfate salts (Na2SO4, CuSO4 + Na2SO4, FeSO4,
Fe2(SO4)3, (NH4)2SO4, and CaSO4) as well as 0.25 mol L−1 EDTA so-
lution were also evaluated as precipitants.
The mixtures were centrifuged at 2380 rpm (for 5–20 min) to ac-
celerate the separation of the precipitate. A 0.5-mL aliquot of the su-
pernatant was transferred to an Eppendorf® tube and mixed with
1.0 mL of 0.25 mol L−1 EDTA solution. Other complexants, i.e., BCA in
an ammonium acetate medium (Rocha & Rocha, 2010) and ammonia
(Fisher & Hall, 1967), were also evaluated for the determination of the
remaining amount of copper.
The photometric measurements were based on the reflected
A.F.S. Silva and F.R.P. Rocha
radiation and conducted directly in the Eppendorf® tubes 3 min after
the addition of EDTA. The intensities of the reflected radiations were
measured under controlled illumination and converted to RGB values.
The values of the R channel were directly used to quantify the proteins
in milk. For the classification of the milk samples, the S values were
calculated by subtracting each measured R value from the reference
value obtained using an Eppendorf® tube filled with water (R mean
value = 208).
Raw milk samples, whose protein concentrations were previously
determined by the NIR reference procedure (AFNOR, 2006), were used
in the optimization step and for accuracy assessment. The optimized
procedure was also applied to raw milk samples containing the pre-
servative, bronopol (0.02% m/v), which exhibits a bactericidal effect.
Because of this preservative, these samples presented an orange color,
whose intensity was measured after protein precipitation by replacing
CuSO4 with 0.25 mol L−1 EDTA in the general procedure. The mean R
values measured at the supernatant (3 ± 1) were subtracted from the
analytical signals, aiming at accurate results.
For the estimation of the limit of detection (LOD), a series of solu-
tions were prepared by the dilution of a milk sample (protein content
3.6% m/v) with water. These solutions were processed by the proposed
procedure (n = 5), and the mean R values were compared to that ob-
tained for the milk sample without dilution (n = 10). The LOD was
determined as the lowest water amount that yielded a mean R value
significantly different from that of the reference sample at a 95% con-
fidence level, as evaluated by the Student t-test.
3. Results and discussion
3.1. General aspects
The proposed procedure is based on the precipitation of milk pro-
teins in a saline medium (salting-out effect), followed by the mea-
surement of the precipitant remaining in the supernatant. The process
also removes other macromolecules dispersed in the milk matrix (Eskin
& Goff, 2013), which are responsible for its turbidity. This minimizes
the light scattering effects in photometric measurements; however, this
is less critical for measurements based on reflected radiation, as in the
present work.
Saline solutions effectively separate proteins because the increase in
the ionic strength hinders the hydrophilic interactions involving these
species. Because the hydrophobic interactions are dominant, the ag-
glomeration of proteins and their removal from the solution are fa-
vored. The efficiency of this process depends on the involved ions as
well as the salt and protein concentrations. In addition, the amino acid
profile, peptidic ligations, and structures of the proteins can affect the
efficiency of the salting-out process (Baldwin, 1996; Leberman, 1991;
Voet, D., Voet, 2010; Wingfield, 1998).
The selection of the precipitant salts was based on the Hofmeister
anion series, PO43− > SO4−2 > CH3COO− > Cl− > Br− >
ClO4− > SCN−, i.e., the most efficient precipitant is PO43− and the
least one is SCN− (Baldwin, 1996; Wingfield, 1998). Because phosphate
ions form insoluble salts (e.g., Cu3(PO4)2), sulfate salts were evaluated
for the proposed procedure. The results were compared to those ob-
tained using EDTA (0.25 mol L−1), diluted acids (0.1 mol L−1 H2SO4 or
HCl), and the joint effect of a salt and a diluted acid, i.e., 0.30 mol L−1
CuSO4 with 0.1 mol L−1 H2SO4. The removal of the milk proteins was
more efficient when CuSO4 was the precipitant, generating a translu-
cent supernatant and a compact pellet after centrifugation. As the dis-
odium salt, EDTA achieves a pH close to the isoelectric point of caseins
(pH = 4.60) and was also efficient for protein precipitation; however, it
led to a colloidal pellet and a comparatively less translucent super-
natant solution. Protein precipitation with the diluted acids was not
efficient because a pH between 3.2 and 3.5 was attained, which favors
the partial resuspension and solubilization of proteins (Voet & Voet,
3
Food Control 115 (2020) 107299
2010). Combination of H2SO4 or Na2SO4 with CuSO4 did not sig-
nificantly improve the protein precipitation.
Copper(II) sulfate was also selected as the precipitant in a procedure
for detecting milk dilutions based on the measurement of the refractive
index of whey (Rezende et al., 2015) utilizing the method proposed by
Ritthausen and Pott in 1873 (Klungsöyr, 1969; Osborne & Leavenworth,
1916). This reagent is also used for protein purification, particularly of
casein, without a significant denaturation of the species (Agarwal &
Gupta, 1994; Osborne & Leavenworth, 1916).
In addition to the promotion of effective salting-out effect by sulfate
ions (Baldwin, 1996; Wingfield, 1998), copper(II) forms complexes
with the side chains of amino acids, mainly histidine, which are
abundant in protein structures (Agarwal & Gupta, 1994). Thus, copper
(II) is removed from a solution proportionally to the amount of the
protein precipitated, which is the basis of the proposed method.
The preferential interaction of copper(II) with amino acids also
ensures that the proposed approach is selective (Agarwal & Gupta,
1994; Ribadeau-Dumas & Grappin, 1989). Moreover, various ligands
are available for the further photometric measurement of copper(II).
3.2. Procedure optimization
The procedure was optimized aiming at quantitative protein pre-
cipitation and minimization of the reagent consumption. The effects of
the CuSO4 concentration (from 0.10 to 0.40 mol L−1) and volumes of
the sample (from 1 to 10 mL) and reagent (from 250 μL to 2 mL) were
evaluated. Quantitative precipitations of the proteins and a translucent
supernatant were achieved with 0.30 mol L−1 CuSO4 (5.00 mL milk and
1.25 mL precipitant solution), in agreement with the precipitant con-
centration previously established (Rezende et al., 2015). This CuSO4
concentration ensures a seven-fold excess in relation to the protein
content (thus favoring precipitation by the salting-out effect) as well as
provides the excess needed for the indirect measurements based on the
remaining amount of the metal ion.
Although the supernatant presented a blue color that is inherent to
the copper(II) aquocomplex, its direct measurement hindered the de-
tection of the adulteration caused by < 15% v/v water. To improve
the detectability, the formation of complexes with EDTA, BCA, or am-
monia was evaluated. The absorption spectra of the corresponding
copper(II) complexes are shown in Fig. 1.
Although BCA yielded a complex with a high molar absorptivity,
reduction of Cu(II) to Cu(I) and a high dilution of the supernatant (ca.
Fig. 1. Absorption spectra of the copper complexes formed with (a) ammonia
(0.1 mol L−1 Cu(II)), (b) BCA (7.5 × 10−4 mol L−1 Cu(I)), and (c) EDTA
(0.1 mol L−1 Cu(II)). Dashed lines delimit the spectral range covered by the R
channel.
A.F.S. Silva and F.R.P. Rocha
400-fold) were needed before the photometric measurements (Rocha &
Rocha, 2010). Moreover, using this reagent is expected to increase the
operational costs and waste volumes, which will reduce the environ-
mental friendliness of the method. EDTA and ammonia presented
comparable results and both can be used as complexants. However,
EDTA was preferred because of the high molar absorptivity of the Cu(II)
complex and the undesirable volatility of ammonia. Moreover, EDTA is
a relatively inexpensive, stable, and widely available ligand. A
0.25 mol L−1 EDTA solution ensured an excess of at least 70% in re-
lation to the remaining amount of copper(II).
Based on the complementarity with the color of the Cu(II)/EDTA
complex, it can be inferred that the spectral range covered by the R
channel overlaps its absorption range (Fig. 1). In fact, the R values
contributed with 70% of the total variation in the RGB values, whereas
the G and B channel contributions were 25% and 5%, respectively.
Because of these aspects and the linear dependence on the protein
concentration (see section 3.3), the intensities of the reflected radia-
tions in the R channel were adopted for the analytical measurements.
3.3. Analytical features
Under the optimized conditions, the proposed procedure presented
a linear response between 0.36% and 3.6% m/v proteins, as described
by the equation: R = (17.9 ± 0.4) C + (68 ± 1), r = 0.999, where R
corresponds to the values of the R-channel and C is the protein content
in % m/v. This concentration range is compatible with the protein
content expected in milk. The LOD was estimated as 0.03% m/v pro-
tein, corresponding to a milk adulteration with as low as 1% v/v water.
This is highly relevant, because most of the approaches for the detection
of milk adulteration are limited to dilutions higher than 10% v/v. The
coefficient of variation was estimated as 3.0% (n = 20). The proposed
procedure demanded ca. 15 min; however, eight samples could be
processed simultaneously, which corresponds to a sample throughput of
ca. 32 assays/h. The procedure consumed ca. 90 mg of each CuSO4 and
EDTA and generated 7.8 mL waste per determination.
Addition of nitrogen compounds aiming to mask the effect of milk
dilution is usual. To evaluate if the proposed procedure was susceptible
to this effect, different substances (whey, melamine, NH4Cl, and urea)
were added along with conducting milk dilution, in concentrations
equivalent to the diminution of proteic nitrogen (Fig. 2). The added
substances did not mask the milk dilution, because all the results were
significantly different from that of the control (unadulterated milk) at a
95% confidence level (Fig. 2, dashed line). Moreover, for a particular
sample dilution, the results obtained with and without the addition of
the nitrogen species were not significantly different, also at a 95%
confidence level. The only exception was the addition of melamine in
the amount needed to compensate dilution with more than 20% v/v
water. This can be explained by the effect of melamine on the internal
structure of casein micelles, which in turn can affect the interactions
between the proteins, water, and CuSO4 (Day, Raynes, Leis, Liu, &
Williams, 2017). Although adulteration with large concentrations of
melamine may interfere in the determination of the protein content in
milk, this is not so for the classification of a non-compliant sample as
compliant. These results demonstrated the selectivity of the proposed
procedure for milk proteins, because of the high stability of the copper
(II) complexes formed with the milk amino acids (protein precipitation
step) and EDTA (color measurement step). Moreover, the addition of
sodium chloride, lactose, and even β-casein did not mask the effect of
the milk dilution, different from that previously observed for protein
determination based on the copper(II)/BCA method (Smith et al.,
1985). In relation to β-casein, the masking effect was not observed,
owing to its low solubility in the medium, arising from its high hy-
drophobicity and the lack of hydration of the commercial product. The
achieved selectivity is a clear advantage versus some approaches for the
detection of milk dilution, such as those based on cryoscopy or re-
fractive index measurements (Rezende et al., 2015).
4
Food Control 115 (2020) 107299
Fig. 2. Analytical signals obtained for different dilutions of a milk sample with
or without addition of masking substances. Error bars indicate the standard
deviations estimated from triplicate measurements and the dashed line corre-
sponds to the control value obtained with unadulterated milk. For each adul-
terant percentage, from left to right, the columns refer to dilution with water,
melamine, whey, ammonium chloride, or urea solutions.
3.4. Applications
3.4.1. Classification of milk samples
The proposed procedure was evaluated as a screening tool of the
compliance of milk samples based on their protein content. To set the
confidence interval for prediction, threshold limits were established
using a milk reference sample, whose protein concentration was de-
termined by the reference procedure (AFNOR, 2006) as 3.5% m/v, and
a solution obtained from the dilution of this reference sample to 3.0%
m/v. These solutions yielded S values (see section 2.3) of 87 ± 4 (3.5%
m/v protein) and 94 ± 3 (for 3.0% m/v protein), n = 9. From the F
and Shapiro–Wilk tests (Mead, Curnow, & Hasted, 2003), it was eval-
uated that the data presented a normal distribution and that the var-
iances were similar at a 95% confidence level. The inferior and superior
limits of the interval were then determined respectively from Equation
(1), which is well established in the procedures of quality control in the
food industry (Shewhart, 1925). The standard error of the mean was
0.5% (calculated by dividing the standard deviation by the square root
of the number of replicates, n = 9), and k was set as 2, corresponding to
a 95% confidence level. Under these conditions, the interval corre-
sponding to a compliant milk sample was established as 84 ≤ S ≤ 98.
(mean ± standard deviation) + standard error of the mean × k (1)
For the validation of the classification model, a set of 29 raw milk
samples whose protein contents (from 3.00 to 3.53%) were determined
by the reference method (AFNOR, 2006) were analyzed in triplicate by
the proposed procedure. Because the S values varied from 86 ± 1 to
90 ± 2, all the samples were classified as compliant, in agreement
with the results obtained by the reference method. Moreover, the
classification approach was applied to five samples from the validation
set after dilution with water from 10% to 40% v/v. Because the S-values
varied from 99 ± 2 to 109 ± 1, these samples were classified as non-
compliant, thus confirming the capability of the procedure to detect
milk dilutions in this range.
The classification approach was also applied to a set of eight com-
mercial milk samples acquired from the local market, associated with
different types of thermal processing, brands, and regions of produc-
tion. As commercial milk samples presented a standardized pattern of
proteins in ca. 3.0% m/v, a specific classification interval was
A.F.S. Silva and F.R.P. Rocha Food Control 115 (2020) 107299

Table 1 CRediT authorship contribution statement

Accuracy assessment of the proposed procedure in comparison to NIR reference
procedure. Mean values and uncertainties of the protein content (% m/v) es- Anna Flavia S. Silva: Conceptualization, Methodology, Validation,
timated from triplicate measurements. Writing - original draft. Fábio R.P. Rocha: Conceptualization, Writing -
Sample Proposed Reference Relative error Estimated F review & editing, Supervision, Funding acquisition.
procedure procedurea (%) valueb
Declaration of competing interest
1 3.79 ± 0.09 3.50 +8.4 6.6
2 3.17 ± 0.09 3.29 −3.6 7.5
3 3.39 ± 0.03 3.29 +3.0 0.8 The authors declare that they have no known competing financial
4 3.33 ± 0.06 3.26 +2.1 3.4 interests or personal relationships that could have appeared to influ-
5 3.4 ± 0.2 3.25 +4.6 37.9
6 3.06 ± 0.07 3.22 −5.0 4.7
ence the work reported in this paper.
7 3.1 ± 0.2 3.20 −3.1 39.1
8 3.0 ± 0.1 3.19 −5.9 9.8 Acknowledgements
a
Coefficient of variation typically of 1.0% (FOSS Analytics). The authors thank the fellowships and financial support from the
b
Critical F value (95% confidence level): 19.0.
Brazilian agencies, São Paulo Research Foundation (FAPESP), National
Council of Technological and Scientific Development (CNPq), processes
established from the solutions containing between 3.0% m/v and 3.2% 424080/2016-8 and 309581/2017-6, and Coordination for the
m/v proteins. For this application, the S values of the compliant sam-
Improvement of Higher Education Personnel (CAPES), financial code
ples should be between 92 and 98, as determined by Eq. (1). By con- 001. This is a contribution of the National Institute of Advanced
sidering this interval, three samples were classified as compliant and
Analytical Sciences and Technology (INCTAA). We also thank Cristiane
four were suspected to be non-compliant. Among the latter, two prob- A. Oliveira and Prof. Paulo F. Machado (from Clínica do Leite, Luiz de
ably presented protein concentrations lower than the threshold limit,
Queiroz College of Agriculture) for supplying milk samples and for
and other two samples probably contained higher than 3.0% m/v helping with the milk analysis by NIR spectroscopy.
protein. Only one sample presented protein content < 3.0% m/v, ac-
cording to the classification criterion. These results demonstrated the References
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number of samples to be analyzed by the reference procedure. green and cost-effective procedure for determination of anionic surfactants in milk
with liquid-liquid microextraction and smartphone-based photometric detection.
Microchemical Journal, 143, 259–263. https://doi.org/10.1016/j.microc.2018.08.
3.4.2. Protein determination 002.
AFNOR Normalisation Norme Française et Internationale « ISO 21543 » depuis.
The proposed procedure was also evaluated for the determination of (décembre 2006). Produits laitiers - Ligne directrices pour l’application de la
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