Characterization of spectral absorbance

for determining the reduction in proteins contents due to heating

(study case of cows milk)
Novia D. Irianti1). Aulia MT. Nasution2)
Photonics Engineering Laboratory
Department of Engineering Physics. FTI ITS
Surabaya 60111. Indonesia
email: 1) wi2ed_ndr@ep.its.ac.id. 2)anasution@ep.its.ac.id

Abstract - Protein is an important component of milk.

Subjecting to heat treatment (e.g. processing or
cooking) will usually cause what so called a
denaturation, i.e. diminishing its content. In this
work a preliminary effort to develop a customized
quantification method based on spectral absorbance
measurement will be devoted. Spectral absorbance of
total milks protein (after subjected to a varied heat
treatment scheme) is measured using a Beckman DU 7500 UV-VIS spectrophotometer. The spectral
derivative technique is then used to analyse the
measured spectral data. The fourth derivative of
absorbance spectra at 290 nm are found to be most
suited for quantifying the progressive changes of
proteins destruction. Comparison is made to
standard Kjeldahl, which shows that the deviation is
in the range of 10 %. This technique is also promising
to be implemented in many biomedical measurements
which are closely related to changes in human bodys
optical properties.
Keyword: milks total protein. denaturation. spectral
absorbance. spectral derivative technique.
1. INTRODUCTION
Milk is a daily food with high protein content that is
important for human health and metabolism, due to
its high nutritional value. As important protein
supplier, protein content in milk needs to be kept at a
useful nutritional levels for human body.
Milk is a kind of emulsion, i.e. colloid globules
surrounding with water-based fluid, and each fat
globule is surrounded by a membrane consisting of
phospholipids and proteins. The largest component in
milk (up to 80 % of total protein) is casein aggregate.
There are four different types of casein, i.e. mainly s1-casein, -s2-casein, -casein, -casein, where most
of them are bound into the micelles. In addition to the
caseins, there are also smaller-structure with a more
water-soluble component. They are known as whey
proteins, which make up to the rest 20 % of the total
protein in milk [1].
Heating treatment on milk is usually used to process
milk to be safely consumable for longer period of

time, reducing the microbial load, as well as

minimizing the risk of food poisoning [2]. When milk
proteins are subjected to thermal processing, the
whey components may undergo a structural change,
commonly known as denaturation. This changes will
be accompanied by structure unfolding as well as
changes in its hydrophobic properties. If its heating
temperatures is above 80oC, the whey proteins will
unfold and interact among themselves and the kcasein to form a heat-induced protein aggregates. At
molecular level, this changes may affect proteins
functionality [3].
Rapid determination of protein content in milk is an
interest for dairy industries and analytical laboratories
for a long time. Total protein concentration in cow's
milk is most commonly determined using the biuret
reaction and Kjeldahl method. These methods are a
bit complicated, time consuming,
and their
measurement accuracy are limited by many
interfering factors.
A more promising approach is offerred by taking
advantage of milks spectral absorption properties in
the UV region (200-380 nm). Under this spectral
band, proteins in milk show a more pronounced
absorbance properties to other non-protein molecules
(e.g. fat). It is then only necessary to dilute the milk
until a negligible level of fat is attainable [4].
The goal of this research is to asses whether is it
possible to measure the total protein content in milk
directly, as well as to detect the proteins reduction
(denaturation) due to heating through its spectral
absorbance measurement.
The succesful of this preliminary effort will be
contibutive for further research to develop customized
techniques for similar measurements. It is also
promising to be implemented in many biomedical
measurements which are closely related to changes in
human bodys optical properties.

2. FUNDAMENTAL THEORIES
2.1 Effect of heat treatment on milk protein
denaturation
Cows milk is mainly composed of water (85 - 88.7 %
of weight), fat (2.4 - 5.5 %), protein (2.3 - 4.4 %).
lactose (3.8 - 5.3 %), minerals (0.53 - 0.80 %) and
organic acids (0.13 - 0.22 %) [l]. Its protein
composition can be classified in two major groups as
presented in Table-1 [5]. The most abundant is
casein, which consists of several components, i.e. the
-s1-casein, -s2-casein, -casein and -casein.
Most of them exist in form of colloidal particles,
known as the casein micelle. The second protein group
is the whey proteins. which include heat-sensitive.
globular. water soluble proteins and enzymes.

Following denaturation, large aggregates will be

formed that unable to cover efficiently the fat droplets
anymore, which leads to emulsions instability. This
behavior can be depicted in Fig. 1, which shows that
the droplet size of the milk protein-stabilized
emulsions do increase proportional to increasing
heating treatment.

Table-1: Protein composition of cows milk [5].

Major milk proteins

Casein

Whey

[gr / l]

-lactalbumin

1.20

-s1-casein

11.60

-s2-casein

3.00

-casein

9.60

-casein

3.60

-casein

1.60

imunoglobulin

0.60

lactoferin

0.30

-lactoglobulin

3.00

albumin serum

0.40

Heating treatments, which commonly regarded as

essential step in dairy product processing, will affect
the rheological as well as structure of its protein
emulsions. It will then determine to a large extent the
consumers acceptability to the milk products. It has
been documented that heating has an impact on the
particle size of model emulsions stabilized with milk
proteins [6].
When milk proteins solutions are subjected to thermal
processing at temperatures around 80 oC or above,
they will exhibit significant loss of emulsifying
ability. The proteins may undergo structural changes.
commonly known as denaturation. Denaturation is a
process in which protein, or nucleic acid in general,
will lose its tertiary, secondary, as well as quartenary
structures. In this case, it will lose the properties of
amino acid structures by dissolution of hydrogen
bonding and other secondary forces that governing the
molecule, which eventually lose many of proteins
biological properties. So the extent of the heat
treatment will proportional to the denaturation level of
milk proteins.

Fig. 1: Emulsion droplet size distribution in fresh milk

stabilized with preheated whey proteins [7]

2.2 Protein interaction with light

The characteristic absorption band of milks protein is
around wavelength of 280 nm, which arises due the
combined oscillator strengths of the lowest energy
transitions of the three aromatic amino acids: i.e. Trp
(Triptophan), Tyr (Tyrosin), and Phe (phenylalanine).
They are the large aromatic residues that are normally
found in the interior of a protein, and are known to
be important for protein stability.
The Tyrosine has special properties since its hydroxyl
side chain may function as a powerful nucleophile in
an enzyme active site and is a common site for
phosphorylation in cell signaling cascades. Meanwhile
the Tryptophan has the largest side chain and is the
least common amino acid in proteins. It has spectral
properties that make it the best inherent probe for
following proteins folding and conformational
changes associated with biochemical processes.
Spectral absorptivity characteristics of these amino
acids is shown in Fig. 2.

Fig 2. Molar absorbtivity of amino acids: Tyrosin,

Triptophan, and Phenilalanin [adapted from 8]

The molar absorptivity for phenylalanine is quite low

in 280nm region, thus it can normally be ignored [8].
[8
Meanwhile due
ue to the lower molecular symmetry of
Trp and Tyr, the molar absorptivities of Trp and Tyr
are much higher than that of Phe,
Phe with respective
maxima of 278 nm and 275 nm [8]. They are to be the
main contributors to the proteins
protein
absorption
spectrum. Separating
arating the characteristic contribution of
these two amino acids can be achieved by
differentiation of the absorption spectrum.
spectrum
The peptide bond absorbs strongly in the far UV
spectrum. This very strong absorption band has been
used in protein determination. As shown in Fig 2. the
strongest absorption band is at 270 290 nm. Because
of interference spectra due to absorption by oxygen as
well as lower detection sensitivity below 200 nm
(vacuum-UV) by most of the available conventional
spectrophotometers. the measurements are more
conveniently started at 210 nm.
2.3 Absorbtion Spectrophotometry
pectrophotometry
Spectral absorbance can be defined as the ratio
between the intensity of radiation absorbed by matter
with the intensity of radiation that comes at a
particular wavelength. The value of light absorption
(absorbance) of atoms or molecules can be expressed
as the
Beer-Lambert Law. Thelaw states that
absorbance of light by a medium is proportional to the
mediums
intrinsic
absoption
characteristics
(absorbtivity), concentration, and light path in the
medium, as written in the following equation:


  .  .    log  


(1)

transmitted light. which is detected by photodetector.

is recorded on computer in spectral basis. The
respected plot of spectral absorbance can be obtained
based on measured data of and  and with the help
of equation (1).
2.4 Spectral Derivative Technique
Spectral derivative technique is in principle based on
mathematical transformation of original spectral curve
(zeroth order) into its respective nth order derivative
spectra.
The technique of spectral derivative analysis was
firstly introduced by Rutherford in the
th 1920s. At that
time he proposed the use of first derivative to enhance
the fine-structure
structure in mass spectrometry.
spectrometry The second
and higher order technique was then patented 30 years
later by two British industrial chemists.
chemist
The renaissance of interest in derivative spectroscopy
was stimulated by the work of Butler and Hopkins in
the mid-1970s. Their work related particularly to UVUV
Vis and fluorescence spectroscopy.
spectroscopy The advancements
of computers and optical detector technologies had
much contributed to the mushrooming exploitation of
the technique in many branch of applications.
applications such as
analytical biochemistry,,
clinical chemistry,
pharmaceuticals, as well as biomedical [10].
[
An
extensive review on the recent developments in
derivative spectrophotometry can be read in [11].
[
Typical form of derivative spectra from its zeroth
order (original absorbance function) until its higher
nth-order
order derivatives can be schematically shown in
Fig. 3.

where:
A is the absorbance
 is the molar absorptivity (L mol-1 cm-1)
 is the molar concentration (mol L-1)
 is the light-path in the material (cm)
and  are the detected
cted and probing light
intensities. repectively
In general. the measurement of spectral absorbance
using absorption spectrophotometry technique can be
shown as follow:

Fig 3. Principle of spectral

ectral absorbance measurement [9]
[9

A wide-spectrum of UV-VIS lightsource after passing

through a monochromator component is subjected to
probe the sample. The probing configuration
normally adopts an in-line arrangement.
arrangement The reduced

Fig 3. Typical form of derivative spectra

from absorbance function

Compared with conventional spectrophotometric

determinations, derivative spectrophometry has

proved to be a great value in eliminating the unwanted

interference signals arises from measurement system.
Such a interfering signals might be represented, i.e.
by baseline and overlapping spectra. So the derivative
spectrophotometry represents an elegant approach to
the problem of resolving overlap spectra in chemical
analysis. It provides much better technique in finding
the hidden fingerprints of analyzed substance in
compare to analysis of original spectral.
Derivative spectra can be obtained by optical,
electronic, or mathematical methods. Optical and
electronic techniques were used on early UV-VIS
spectrophotometers, but nowadays it has largely
superseded by mathematical techniques [10]. The
advantages of the mathematical techniques are that
derivative spectra may easily be calculated. Spectral
absorbance
function can be approximated by
Gaussian fitting of the curve, so that the absorbance
values contained in the spectral graph can be fitted by
these curves. Derivative functions can then being
derived from these fitting functions.
3. MATERIALS AND METHODS
3.1 Materials and samples
The samples of fresh cow's milk were collected during
April 2010 from a cows farm in Jemursari-Surabaya,
and a bottle of distilled water (H2O) also required for
samples dilution was also prepared.
3.2 Samples treatment
Dillution
The only big molecules in milk are fat and protein,
which can cause obvious light absorbtion. The milk
samples must be diluted to a concentration that is well
within the detectability range of the instrument. A
dilution of 100 - 500 times with distilled water (H2O)
prior to measurement is suggestible, since the
spectrophotometer can only read
transparent and
completely dissolved samples. In addition. the dilution
itself will reduce the excessive fat content in the
sample.
Heating
Heating treatment of the samples was done to simulate
the degree of protein destruction due to heating. This
task was accomplished by using a Magnetic Stirrer,
which was set to a certain temperature and stirrer
speed. Samples was subjected to a heating scheme
from 80C, 90C, 100C, and 110C (each at heating
duration of 100s, 300s, and 1000s ).
Homogenization
The raw milk sample was homogenized by a magnetic
stirrer devices at 2 rpm speed of stirring. The
preprocessed samples that used for absorbance
measurement are ones that have been separated from
the sediments, and hold into transparent cuvettes for
measurement.

The average diameter of the protein in the milk after

homogenization is 120 nm . Other particles (lactose
and inorganic salts) are dissolved in milk, and their
diameters are much less than that of protein.
Therefore, the measured absorbance of UV light
source can accurately determine the content of the
protein in the milk.
3.3 Equipments
In this study, the Beckman DU-7500 UV-Visible
spectrophotometer was used to obtain the spectral
absorbance of samples. Range of scanning
wavelengths are 200 - 380 nm with a scan rate of 100
nm/min. Besides, some additional equipments are also
used, i.e. quartz cuvetes to hold samples, filter papers,
bekker glasses, pipettes, erlenmeyer flasks, and
measuring flasks.
3.4 Benchmarking
For the purpose of referencing, absolute protein
content (in mg/mg of milk) from the diluted milk
sample (prior to subjection to heating scheme) was
measured using Kjeldahl method. This measurement
result was utilized for calculating the proteins
progressive destruction due to heating scheme. which
was relatively measured based spectral analysis of the
absorbance data.
Meanwhile similar measurements were also done
for samples after underwent heating scheme
treatments. These data are used to asses the accuracy
of proteins contents determination using proposed
spectral absorbance method
3.5 Spectral analysis
To compute the derivative spectra, the software
package MATHCAD 14 was used. Derivative spectra
were calculated from measured absorbance spectra by
approximating them using a Gaussian fitted functions
(generated in MATLAB-7.1). The amplitudes of the
signatures peaks and troughs were obtained directly
from the derivative spectra. Spectral band which
shows following criteria, i.e. regularly pattern of
changes in accordance to variability of heating
scheme, no shifting, and symmetrical is used for the
purposed quantification.
4.

RESULTS AND DISCUSSION

4.1 Measurement results

The measurements were carried out in the wavelength
range of 200380 nm, corresponding to the
absorbance of protein molecules and aromatic amino
acid. Typical measurement absorbance spectra
obtained using the spectrophotometer is shown in Fig
5. The graph clearly indicates that the change of
heating scheme will deminish the protein absorbance.

nm is used to quantify the total protein content. This

makes it possible to use specific wavelength at 290 nm
trough over the peak to quantify the protein content in
milk with various heating treatment.
4.3 Effect of heating treatment of milk on protein
content reduction
Thermal processing affects the functionality of the
ingredients used for emulsion formation in milk
solution, which among others is the diameter of the
protein, which has a close relationship with protein
content in milk. When milk is heated, the change in
particle size due to destruction of protein aggregate is
dependent on the temperature and duration of heat
treatment. An increase in temperature will increase
the percentage of protein content reduction. Fig 8
shows the percentage of protein content in milk
sample due to heating treatment, i.e. temperature and
heating time.

Fig 5: Typical plot of spectral absorbance from sample

with a) various temperature and b) various
heating time at T=80C

4.2 Spectral derivative of protein

A higher-order derivative spectra will improve
the resolution of complex spectra at the value of
decreasing the amplitude. As can be seen in Fig.6,
results indicates that the derivation in fourth order will
show best trend in diminishing magnitude of the
spectra, in accordance with changes in heating
scheme. The magnitude of the spectra which are
centered at 290 nm will dimisnish as the degree of
protein destruction increases. In other words, as the
heating scheme increases such that more proteins
molecules destructed, less protein will absorb the
probing light, which means lower absorbance

Fig 8: The percentage of protein content

in milk sample due to heating treatment.

The figure shows that the highest heating scheme (i.e.

temperature of 110oC and 1000s heating time), it will
reduce the protein content ca. 96.3%.
4.4 Protein content of milk samples
To ascertain whether the fourth derivative method
might be an alternative for direct determination of
protein milk samples, a comparison was made
between protein contents measured by this method and
by the reference method (Kjeldahl method). The range
of total protein content for cow's milk samples
determined are listed in Table 1.
Table 1: Comparison of milks total protein determination
by derivative spectrophotometry vs Kjeldahl

Fig 6. Fourth derivative spectral absorbance

of milk sample in 90oC

of protein content in the sample. The changes in

magnitude of derivative spectral at 290 nm is
significant, therefore the amplitude of the peak at 290

Treatment
scheme
w/o heating
80 C. 10 s
90 C. 10 s
100 C. 10 s
110 C. 10 s

Kjeldahl
0.0261
0.0215
0.0093
0.0096
0.0042

Derivative Spectra
analysis
0.0261
0.0221
0.0121
0.0085
0.0038

The comparison in the table above can be visualized in

the Fig 7 below.

[7]

[8]

[9]
Fig 7: Graph of comparison of milks total protein
determination by derivative spectrophotometry vs Kjeldahl
The values of protein concentration of milk samples
determined based on derivative spectral method were
not significantly different from the ones obtained by
measurement using Kjeldahl method.
5.

CONCLUSION

The fourth derivative of UV spectra in 200-380 nm

wavelength range can be used to determine the
protein content in milk.
Measurement of protein content is done based on
thr magnitude of the derivative spectra centered at
290 nm.
Protein denaturation is already happened at
temperature 80oC, and the largest protein
denaturation occurs at temperature 110oC with
heating time 1000s, where it decrease untill
96.3%. The remaining protein concentration was
0.29 gr/ml

[1]

[2]

[3]

[4]

[5]
[6]

REFERENCES
McGee. H., 1984, Milk and Dairy Products,
On Food and Cooking: The Science and Lore of
the Kitchen, Charles Scribners Sons, New
York, p. 353.
McKinnon. I. R., Yap. S. E., Augustin. M. A.,
and Hermar. Y., 2009,
Diffusing-wave
spectroscopy
investigation
of
heated
reconstituted skim milks containing calcium
chloride, Food Hydrocolloids, 23, 11271133.
Singh, H. and Creamer, L. K., 1992, Heat
stability of milk, in Advanced Dairy Chemistry
(Ed. P. F. Fox), Vol. 1 Proteins. (pp.621656).
Springer, London.
S.J. Rowland, 1938, The Determination of the
Nitrogen Distribution in Milk, J. Dairy Res. 9,
42.
Walstra, P. and Jenness, R.,1984, Dairy
Chemistry and Physics, Wiley, New York.
McSweeney. S. L.. Mulhivill. D. M.. and
OCallaghan. D. M.. 2004. The influence of pH
on the heat-induced aggregation of model milk
protein ingredient systems and model infant
formula emulsions stabilized by milk protein
ingredients. Food Hydrocolloids. 18. 109125.

[10]

[11]

Millqvist-Fureby. A. M.. Elofsson. U.. &

Bergenstl. B., 2001, Surface composition of
spray-dried milk protein-stabilised emulsions in
relation to pre-heat treatment of proteins.
Colloids and Surfaces B: Biointerfaces. 21. 47
58.
Wetlaufer. D. B., 1962, Ultraviolet spectra of
proteins and amino acids. Adv. Protein Chem.
17. 303-390.
Taiz. L. and Zeiger. E., 2006, Plant Physiology,
4th Ed., Lincoln Taiz and Eduardo Zeiger,
Sinauer Assc. Inc. Publ.Sunderland. MA-USA.
Fell. A.F.. 1983, Biomedical applications of
derivative spectroscopy, trends in analytical
chemistry. vol 2(3).
Rojas. F.S. and Ojeda. C.B., 2004, Recent
development in derivative spectrophotometry:
2004 2008: A Review, Analytica Chimica
Acta, 635, p22-44.