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A non-protein nitrogen index for discriminating raw

milk protein adulteration via the Kjeldahl method
Cite this: Anal. Methods, 2015, 7, 9166
Peipei Gao,a Zhicheng Li,*a Linsen Zan,b Tianli Yuea and Bo Shia

Received 3rd June 2015
Accepted 10th September 2015
DOI: 10.1039/c5ay01422k
www.rsc.org/methods
In this paper, we have developed the concept of the non-protein nitrogen (NPN) index defined as a ratio
coefficient of NPN content and crude protein nitrogen (CPN) content in milk. To demonstrate the NPN
index, the NPN and CPN in a series of milk samples were determined by using the Kjeldahl method and
the trichloroacetic acid precipitation method (TCA). The NPN index of raw milk was chosen for
accurately discriminating milk adulteration with ammonium chloride, ammonium sulfate, urea,
melamine, and whey powder. The results showed that all of the NPN indices of adulterated milk fell
outside the discriminating ranges, which were supposed to be from 0.8084 to 1.4987, unless the amount
of melamine was less than 0.2 mg kg
1 in adulterated milk. All the results suggested that the NPN index
could be used to accurately detect milk adulteration with nitrogen-rich substances and provided an
efficient way to monitor milk quality.

consumption of milk and other dairy products in China

Introduction
Milk is one of many primary sources of nutrition for infants and
patients due to its unique nutritional value. Milk protein
contains a vast variety of amino acids, which not only promote
one's growth and development but also maintain one's health.
Casein and whey protein are two major proteins in milk.1,2 A
high concentration of true protein (TP) in milk is highly desir-
able to satisfy the dairy industry demand.3 TP can be dened as
the protein corresponding to the protein nitrogen, except non-
protein nitrogen in milk. The TP content in milk is naturally
less than the crude protein content under normal circum-
stances.4 The nitrogenous sources in milk can be divided into
three broad fractions, including casein, whey protein, and non-
protein nitrogen (NPN).5 The NPN (ammonia, urea, creatine,
free amino acids and so on) constitutes 5% (w/w) of total
nitrogenous compounds in milk.6 As a protein-rich substance,
milk is widely supplied in the diet. During the past decades,
with the emergence of protein adulteration in milk, the NPN
fraction of milk has been gaining attention.
Milk adulteration is a general and serious concern in the
dairy industry all over the world.7 The adulterated milk not only
reduces the nutritive value of dairy products but also leads to
food security crises, such as the melamine milk scandal which
occurred in China in 2008 which caused the fatality of six
infants and over 51 900 infants and young children to be sick
with serious kidney complications.8 Following this incident, the
a
College of Food Science and Engineering, Northwest A&F University, Yangling,
Shaanxi, 712100, PR China. E-mail: lizhicheng@nwsuaf.edu.cn; Fax: +86-29-
87092486; Tel: +86-29-87092486
b
College of Animal Science and Technology, Northwest A&F University, Yangling,
Shaanxi, PR China
suffered a major decline.9 As a consequence, milk adulteration
has impacted long-term benets for Chinese farmers, dairy
industries and consumers. Therefore, an efficient and conve-
nient method is urgently needed to evaluate the protein quality
of raw milk.
The current analytical techniques for detecting melamine, or
other harmful substances in milk are HPLC, LC-MS/MS, GC-MS,
and GC-MS/MS as well as different MS.10–12 These techniques are
rapid, sensitive and automatic,10 especially present the chem-
ical structure information of NPN adulteration reagents.11,12 The
newly developed methods for detecting pure protein content in
milk or food are moving reaction boundary electrophoretic
titration (MRBET),13,14 visual moving reaction boundary titra-
tion (MRBT)15 and microuidic MRBET.16 They are simple,
rapid, sensitive and of low cost, particularly avoid the obvious
inuence of NPN, and have potential use in quality control of
food and milk. Currently, the standard technique used for
protein content quantitation of milk or food is still the Kjeldahl
method,17 which is used to calculate the crude protein content
rather than the true protein content from the nitrogen content
of milk or food, and cannot be used for adulterated milk and
food.18
Recently, Finete et al.19 combined the Kjeldahl method with
spectrophotometric methods for screening milk adulteration
(melamine, ammonium sulphate, or urea). This year, Liu et al.20
have developed the idea of index Q for effectively discriminating
articially adulterated milk from unadulterated milk. During a
run of index Q, both normal and adulterated samples of bovine
raw milk were determined via a Fourier transform infrared
spectroscopic instrument, focusing on the traditional indices of
quality, such as fat (FAT), protein (PRO), lactose (LAC), total

9166 | Anal. Methods, 2015, 7, 9166–9170 This journal is © The Royal Society of Chemistry 2015

Paper
solids (TS), non-fat solid (NFS), freezing point (FP) and somatic
cell counts (SCC). Then the parameter pairs, such as TS-FAT and
FP-LAC, were chosen as index Q for accurately discriminating
milk adulteration with maltodextrin and water, and with nine
other kinds of synthetic adulterants. However, the idea of a non-
protein nitrogen index has not been proposed so far, which has
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potential use in control of raw milk quality.
Thus, herein we have dened the concept of the non-protein
nitrogen index as the ratio between NPN and crude protein
nitrogen (CPN), performed the relevant experiments demon-
strating the validity of the non-protein nitrogen index, and
nally used the index for successfully evaluating the protein
quality of raw milk.
Materials and methods
Samples and reagents
Ten batches of 126 milk samples were collected from 4 repre-
sentative milk stations (Shaanxi, China) in a year. Every batch
contained milk samples from the 4 different stations (marked
as A, B, C and D) collected at different times and every batch
contained 3 (in a few cases 4) milk samples from each milk
station. All reagents were of analytical grade purity in this
research.
Adulterated milk samples were prepared by adding adulter-
ants into raw milk, containing ammonium chloride, urea,
ammonium sulfate, and whey powder. The added amount for
every adulterant was chosen such that it improves 0.2% (w/w)
protein content in raw milk. Each kind of adulterated sample
was prepared in triplicate, each group having three samples to
be tested, and the nal result was obtained using the mean
value of each group. The adulterated milk samples of melamine
were prepared at concentrations of 2000, 200, 20, 2 and 0.2 mg
kg
1 (n ¼ 3 at each concentration), respectively.
The adulterated milk samples were stirred until adulterated
substances dissolved completely at room temperature.
Sample CPN analysis by the Kjeldahl method
Unadulterated raw milk samples and the adulterated ones were
analyzed to determine the CPN by using the Kjeldahl method
with modications. The raw milk samples (5.000 g) were
digested with 25 mL of sulfuric acid by using a controlled-
temperature furnace. When the initial temperature of the
controlled-temperature furnace reached 200  C, a gradient
temperature was used to heat up 100  C every half-hour until it
reached 450  C, and then this temperature was maintained to
clear the digestion solution. The nitrogen value conversion into
protein using the factor of 6.38. The rest of the experimental
processes were carried out at temperatures of 20  C–25  C. The
CPN was calculated using formula (1).21
1:4007  ðVs
Vb Þ  M
Nitrogen; % ¼ (1)
W there was protein adulteration in milk. Although each raw milk
where Vs and Vb ¼ mL HCl titrant used for the test portion and
blank, respectively; M ¼ the molarity of HCl solution; and W ¼
the test portion weight, g.
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Analytical Methods
Sample NPN analysis by the TCA precipitation method
The unadulterated raw milk samples and the adulterated milk
samples were analyzed to determine the NPN by using the tri-
chloroacetic acid precipitation method.22 In order to precipitate
the milk protein, 150 g L
1 trichloroacetic acid was added into a
prepared milk sample. Non-protein nitrogen was present in the
supernatant uid, a certain amount of ltrate aer digestion
and distillation, and titrated using 0.01 mol L
1 hydrochloric
acid solution. The NPN was calculated using formula (2).22
1:4007ðVs
Vb ÞMr
wnp ¼  (2)
mf mm ðmt
0:065mm Þ
where wnp ¼ the non-protein-nitrogen (NPN) content, expressed
as a percentage of mass; Vs and Vb ¼ mL HCl titrant used for the
test portion and blank, respectively; Mr ¼ molarity of HCl
solution: mm ¼ the test portion weight, g; mt ¼ the test portion
with an addition of 40 mL of trichloroacetic acid solution
weight, g: mf ¼ the weight of ltrate used to digest, g; and 0.065
¼ the multiplication factor.
All determinations were done in triplicate independent
samples.
Calculation of the NPN index
The true protein nitrogen was dened as TPN ¼ CPN
NPN.
The corresponding true protein was dened as TP ¼ (CPN

NPN)  6.38. The NPN index was calculated as NPN/CPN  100.
Data analysis
The statistical analysis was carried out by using SPSS 17.0
soware. The data were expressed as mean  standard devia-
tions (SD).
Results and discussion
Analysis of the raw milk samples
Table 1 presents the content of the NPN in total nitrogen,
ranging from 0.8% to 1.5% (w/w). There were some samples
which had high CPN contents but the corresponding NPN
contents were low. Others had low CPN content, while the
corresponding NPN was high, this mainly depended on the
composition of the milk samples. The average TP content of the
raw milk samples was 3.06  0.16% (w/w), which was consistent
with previous research.23
The NPN index model
The NPN indices of the raw milk (10 batches) were distributed
within a range. The data are represented by dots between the
limit lines, the upper bound was 1.4987 and the lower bound
was 0.8084 (Fig. 1). These indices were chosen as a NPN index
model, the range within the scope indicated that the milk was
normal. When the range was not in this scope, it demonstrated
sample contains different proteins and non-proteins, the
content was basically distributed within a range. By using this
feature, the non-protein nitrogen indices of raw milk were
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Analytical Methods Paper

Table 1 The protein content and the non-protein nitrogen content of raw milk from different milk stations (%)

CPN TPN NPN CPN TPN NPN

Number A B

1 0.487  0.003 0.480  0.003 0.00730  0.00035 0.499  0.004 0.492  0.004 0.00696  0.00004
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2 0.462  0.003 0.456  0.003 0.00602  0.00013 0.443  0.013 0.437  0.013 0.00648  0.00011
3 0.475  0.011 0.469  0.011 0.00659  0.00011 0.516  0.006 0.510  0.006 0.00557  0.00018
4 0.442  0.003 0.436  0.003 0.00584  0.00015 0.494  0.006 0.488  0.006 0.00574  0.00028
5 0.454  0.011 0.449  0.011 0.00454  0.00038 0.503  0.012 0.496  0.012 0.00672  0.00003
6 0.444  0.021 0.439  0.021 0.00543  0.00032 0.514  0.011 0.508  0.011 0.00587  0.00012
7 0.508  0.011 0.504  0.011 0.00436  0.00037 0.496  0.009 0.491  0.009 0.00493  0.00015
8 0.507  0.019 0.502  0.019 0.00563  0.00039 0.528  0.008 0.521  0.008 0.00708  0.00025
9 0.463  0.018 0.456  0.018 0.00647  0.00014 0.486  0.014 0.480  0.014 0.00615  0.00031
10 0.453  0.018 0.448  0.018 0.00542  0.00040 0.470  0.015 0.464  0.015 0.00589  0.00027

Number C D

1 0.522  0.002 0.518  0.002 0.00422  0.00033 0.485  0.025 0.480  0.025 0.00493  0.00033
2 0.490  0.018 0.485  0.018 0.00485  0.00037 0.468  0.024 0.463  0.024 0.00559  0.00018
3 0.485  0.020 0.480  0.020 0.00454  0.00019 0.467  0.017 0.461  0.017 0.00637  0.00026
4 0.462  0.008 0.457  0.008 0.00488  0.00011 0.517  0.003 0.509  0.003 0.00759  0.00012
5 0.469  0.011 0.464  0.011 0.00571  0.00027 0.473  0.014 0.467  0.014 0.00554  0.00029
6 0.527  0.025 0.520  0.025 0.00719  0.00013 0.470  0.017 0.463  0.017 0.00637  0.00016
7 0.486  0.015 0.479  0.015 0.00661  0.00057 0.445  0.016 0.441  0.016 0.00361  0.00032
8 0.461  0.016 0.455  0.016 0.00584  0.00046 0.465  0.020 0.459  0.020 0.00584  0.00013
9 0.469  0.011 0.462  0.011 0.00691  0.00052 0.478  0.029 0.472  0.029 0.00667  0.00022
10 0.457  0.020 0.451  0.020 0.00589  0.00048 0.527  0.033 0.520  0.033 0.00718  0.00028

calculated and they uctuated within a certain range. In other
words, the non-protein nitrogen index of the normal milk
should be within the scope. If milk was adulterated with
melamine, or other harmful substances, it will not comply with
this variation. Therefore, the establishment of this model has a
certain signicance.
Verication of the NPN index model
The adulterated milk samples were prepared to verify the NPN
index model and the results displayed a fairly high level of
sensitivity. When the raw milk samples were added with
ammonium chloride, its corresponding NPN indices were
5.0549, 5.2054, and 5.4249, respectively, which were far greater
than the model's upper bound 1.4987, as shown in Fig. 2.
Meanwhile, the NPN index of adulterated milk with ammonium
sulfate and urea had the same results. However, the NPN index
of raw milk combined with whey powder was under the lower
bound of the model. The US Food and Drug Administration
(FDA) set the melamine MRL at 0.25 mg kg
1 in milk.24 In this
study, the concentrations of the melamine adulteration in the
raw milk ranged from 2 g kg
1 to 0.2 mg kg
1. The

Fig. 1 Construction of model with the NPN index of raw milk. (A) the NPN index (n ¼ 40).

9168 | Anal. Methods, 2015, 7, 9166–9170 This journal is © The Royal Society of Chemistry 2015

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Fig. 2 Verification the NPN index model with the adulterated milk samples and the milk added whey powder samples. (,) ammonium chloride,
(B) ammonium sulfate, (:) urea, (-) whey powder, and (*) melamine.
corresponding NPN indices ranged from 2.6343 to 1.5956, and
the model could not be used to identify milk with melamine less
than 0.2 mg kg
1.
The discrimination accuracy of the model could be up to
100% under the conditions of adding ammonium chloride,
ammonium sulfate, whey powder, or urea to the raw milk to
improve 0.2% (w/w) protein content, or the milk mixed amount
of melamine is more than or equal to 0.2 mg kg
1. Liu et al.20
built the model of index Q with a range of normal milk
measurement values from
0.0038 to 0.0036 to differentiate
articially adulterated milk from unadulterated milk. The
indices were obtained by linear regression analysis and residual
analysis. Comparing this method with that reported by Liu
et al.,20 we found that the merits of this method were that it was
more convenient and could avoid complex analysis processes.
The present study was to calculate the NPN indices by
measuring the nitrogen content of raw milk and these indices
were used to evaluate the quality of milk protein. Although it
took a long time to digest protein during the test to gain CPN
and NPN, the good news was that nitrogen determination can
now be more rapidly carried out by using an FP528 Automatic
Nitrogen Analyser.25–27 Meanwhile, spectroscopic methods28,29
and chromatographic methods30 can be used to determine the
nitrogen content, which will be more advantageous to deter-
mine the NPN index. The main advantages of the NPN index
model are as follows: (1) it can determine whether or not there
are mixed adulterated nitrogenous substances in milk, (2) it can
avoid testing single nitrogen-rich substances one at a time,
especially when we have to determine whether milk samples
contain protein adulteration. The limitation of this model is
that only the non-protein nitrogen indices of 126 raw milk
samples were measured, which were produced by Holstein cows
of the 4 major regions in Shaanxi province, in China. The non-
protein nitrogen index model in this study needs to be applied
to other milk-producing countries and regions, so that the test
method can be more convincing and credible.
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Analytical Methods
Conclusions
This study explored the foundation and application of the non-
protein nitrogen (NPN) index to evaluate the protein quality of
raw milk. The NPN index is a ratio coefficient of NPN and CPN
content in raw milk. The NPN index of unadulterated milk
ranges from 0.8084 to 1.4987, which was veried to be capable
to detect non-protein nitrogen adulteration (ammonium chlo-
ride, urea, ammonium sulfate, and melamine) and protein
nitrogen adulteration (whey powder) in raw milk. The signi-
cance of this work was that the protein quality of raw milk can
be evaluated using the simplied method, which has great
potential in the verication of protein adulteration in raw milk.
Acknowledgements
We thank the Ministry of Science and Technology (MOST),
Government of PR China (No. 2012BAD12B07), and the
Department of Science and Technology of Shaanxi Province
(2011KTCL02-11) for their nancial support.
References
1 Y. W. Parka, M. Juárez, M. Ramos and G. F. W. Haenleind,
Small Ruminant Res., 2007, 68, 88–113.
2 A. M. Michaelidou, Small Ruminant Res., 2008, 79, 42–50.
3 A. A. Aquino, Y. V. R. Lima, B. G. Botaro, C. S. S. Alberto,
K. C. Peixoto Jr and M. V. Santos, Anim. Feed Sci. Technol.,
2008, 140, 191–198.
4 E. J. Depeters and J. D. Ferguson, J. Dairy Sci., 1992, 75, 3192–
3209.
5 S. J. Rowland, J. Dairy Res., 1938, 9, 47.
6 M. Olalla, M. Ruiz-López, M. Navarro, R. Artacho, C. Cabrera,
R. Giménez, C. Rodriguez and R. Mingorance, Food Chem.,
2009, 113, 835–838.
7 S. Das, M. Sivaramakrishna, K. Biswas and B. Goswami, Sens.
Actuators, A, 2011, 167, 273–278.
Anal. Methods, 2015, 7, 9166–9170 | 9169

Analytical Methods
8 X. M. Xu, Y. P. Ren, Y. Zhu, Z. X. Cai, J. L. Han, B. F. Huang
and Y. Zhu, Anal. Chim. Acta, 2009, 650, 39–43.
9 C. P. Zhang, J. F. Bai, B. T. Lohmar and J. K. Huang, China
Econ. Rev., 2010, 21, S45–S54.
10 L. G. Zhang, X. Zhang, L. J. Ni, Z. B. Xue, X. Gu and
S. X. Huang, Food Chem., 2014, 145, 342–348.
Published on 15 September 2015. Downloaded by KUNGL TEKNISKA HOGSKOLAN on 29/11/2015 10:03:49.
11 J. S. Garcia, G. B. Sanvido, S. A. Saraiva, J. J. Zacca,
R. G. Cosso and M. N. Eberlin, Food Chem., 2012, 131, 722–
726.
12 T. M. C. Motta, R. B. Hoff, F. Barreto, R. B. S. Andrade,
D. M. Lorenzini, L. Z. Meneghini and T. M. Pizzolato,
Talanta, 2014, 120, 498–505.
13 H. Y. Wang, C. Y. Guo, C. G. Guo, L. Y. Fan, L. Zhang and
C. X. Cao, Anal. Chim. Acta, 2013, 774, 92–99.
14 C. Y. Guo, H. Y. Wang, X. P. Liu, L. Y. Fan, L. Zhang and
C. X. Cao, Electrophoresis, 2013, 34, 1343–1351.
15 C. Y. Guo, H. Y. Wang, L. Zhang, L. Y. Fan and C. X. Cao,
Chin. J. Chromatogr., 2013, 31, 1064–1070.
16 H. Y. Wang, Y. T. Shi, J. Yan, J. Y. Dong, S. Li, H. Xiao,
H. Y. Xie, L. Y. Fan and C. X. Cao, Anal. Chem., 2014, 86,
2888–2894.
17 N. K. K. Kamizake, M. M. Goncalves, C. T. B. V. Zaia and
D. A. M. Zaia, J. Food Compos. Anal., 2003, 16, 507–516.
18 B. M. Balabin and S. V. Smirnov, Talanta, 2011, 85, 562–568.
9170 | Anal. Methods, 2015, 7, 9166–9170
View Article Online
Paper
19 V. L. M. Finete, M. M. Gouvêa, F. F. C. Marques and
A. D. P. Netto, Food Chem., 2013, 141, 3649–3655.
20 J. Liu, J. Ren, Z. M. Liu and B. H. Guo, Food Chem., 2015, 172,
251–256.
21 AOAC, AOAC Official Method 991.20, rst action 1991 and
nal action 1994.
22 ISO, IDF 020-4-2001, International Organization for
Standardization and International Dairy Federation:
Geneva, Switzerland, 2001.
23 A. Melfsen, E. Hartung and A. Haeussermann, Biosyst. Eng.,
2012, 112, 210–217.
24 A. Filazi, U. T. Sireli, H. Ekici and A. Karagoz, J. Dairy Sci.,
2012, 95, 602–608.
25 R. L. Lindroth, T. L. Osier, H. R. H. Barnhill and S. A. Wood,
Biochem. Syst. Ecol., 2002, 30, 297–307.
26 M. W. C. D. Palliyeguru, S. P. Rose and A. M. Mackenzie, Br.
Poult. Sci., 2011, 52, 359–367.
27 A. Doyen, L. Saucier, L. Beaulieu, Y. Pouliot and L. Bazinet,
Food Chem., 2012, 132, 1177–1184.
28 R. Tsenkova, S. Atanassova, Y. Ozaki, K. Toyoda and K. Itoh,
Int. Dairy J., 2001, 11, 779–783.
29 A. Bogomolov, S. Dietrich, B. Boldrini and R. W. Kessler,
Food Chem., 2012, 134, 412–418.
30 A. I. Pavlova, D. S. Dobrev and P. G. Ivanova, Fuel, 2009, 88,
27–30.
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