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Food Bioprocess Technol (2011) 4:364–386

DOI 10.1007/s11947-010-0370-0

REVIEW PAPER

Fluorescence Spectroscopy Measurement for Quality


Assessment of Food Systems—a Review
Romdhane Karoui & Christophe Blecker

Received: 13 March 2010 / Accepted: 26 April 2010 / Published online: 12 May 2010
# Springer Science+Business Media, LLC 2010

Abstract The present review gives an overview of the use appropriate analytical tools to investigate food. Fluores-
of fluorescence spectroscopy (i.e., conventional, excitation– cence spectroscopy is an analytical technique whose theory
emission matrix, and synchronous fluorescence) for deter- and methodology have been extensively exploited for
mining changes in food products and their quality during studies of molecular structure and function in the discipline
technological process and storage. From the present review, of chemistry and biochemistry (Strasburg and Ludescher
it was shown that fluorescence spectroscopy is able to 1995). Even though fluorescence is one of the oldest
determine several properties (functional, composition, nutri- analytical methods used (Valeur and Bochon 2001), it has
tional) without the use of chemical reagents. This is due to just, recently, become quite popular as a tool in biological
the use of chemometric tools (descriptive and predictive science related to food technology. An indication for this
methods). The review focuses on the use of fluorescence popularity is the increasing number of research publications
spectroscopy for the determination of the quality of animal about fluorescence as well as the introduction of new
(i.e., dairy, meat, fish, and egg) and vegetable (oils, cereal, commercially available instruments for fluorescence analy-
sugar, fruit, and vegetable) products as well as the identifi- sis, particularly, front-face fluorescence spectroscopy
cation of bacteria of agro-alimentary interest. (FFFS). Traditional right angle fluorescence spectroscopic
technique cannot be applied to thick substances due to large
Keywords Fluorescence spectroscopy . Food systems . absorbance and scattering of light. Indeed, when the
Quality . Chemometrics absorbance of the sample exceeds 0.1, emission and
excitation spectra are both decreased and excitation spectra
are distorted (Karoui et al. 2003). To avoid these problems,
Introduction a dilution of samples (when it is possible) was performed so
that their total absorbance would be less than 0.1. However,
Public interest in food quality and production has increased the results obtained on diluted solution of food samples
in recent decades, probably related to changes in eating cannot be extrapolated to native concentrated samples since
habits, consumer behavior, and the development and the organization of the food matrix is lost. To reply with
increased industrialization of the food supplying chains this request, FFFS could be utilized. The use of only
(Christensen et al. 2006). The demand for high quality and excitation and emission wavelengths could limit the ability
safety in food production obviously calls for high standards of fluorescence spectroscopy to determine the quality of
for quality and process control, which in turn requires food systems. To comply with this requirement, the
variation in the excitation and emission wavelengths allows
simultaneous determination of compounds in several food-
R. Karoui (*) : C. Blecker stuffs. Therefore, it would be interesting to use for each
Department of Food Technology, Gembloux Agro-Bio Tech, food product different excitation wavelengths simulta-
University of Liège,
neously. This could be realized by using synchronous
Passage des Déportés, 2,
5030 Gembloux, Belgium fluorescence spectroscopy (SFS) which presents two inter-
e-mail: karouiromdhane@yahoo.fr esting advantages from our point of view: it (1) allows the
Food Bioprocess Technol (2011) 4:364–386 365

consideration of the whole fluorescence landscape, i.e., fluorescence emission does not only occur at one single
spectra recorded at different offsets and (2) retains wavelength, but rather over a distribution of wavelengths
information related to several fluorophores compared to a corresponding to several vibrational transitions as compo-
classical emission spectrum, which is mainly specific to a nents of a single electronic transition. This is why
sole fluorophore. Synchronous fluorescence spectra are excitation and emission spectra are obtained to describe
obtained by simultaneously scanning both the excitation the detailed fluorescence characteristics of molecules. In
and emission monochromators keeping a fixed wavelength fact, fluorescence is characterized by two wavelength
interval, named offset between them. It gives a narrower parameters that significantly improve the specificity of the
and simpler spectrum. For the SFS technique, the selection method, compared to spectroscopic techniques based only
of a wavelength interval is one of the most important on absorption.
experimental parameter and the parameter should be
optimized, which is carried out by measuring the spectra Quantum Yield (Efficiency)
at various offsets.
This review will provide the reader with the basic Each molecule presents a specific property, which is
principles of fluorescence including the use of this described by number, named quantum yield or quantum
technique, especially the use of the most common FFFS efficiency (  ).
and synchronous fluorescence for the assessment of the
quality of several food systems that will be discussed in number of quanta emitted
f¼ ¼ quantum yield ð1Þ
detail (Table 1). number of quanta absorbed

As illustrated in Eq. 1, the higher the value of  , the


Fluorescence Spectroscopy greater the fluorescence of a compound (e.g., chlorophyll).
A practically non-fluorescent molecule (e.g., carotenoids) is
Definition one whose quantum efficiency is zero or so close to zero
that the fluorescence is not measurable. All energy
Fluorescence is the emission of light subsequent to absorbed by such a molecule is rapidly lost by collision
absorption of ultraviolet or visible light of a fluorescent deactivation.
molecule or substructure, called a fluorophore. Thus, the
fluorophore absorbs energy in the form of light at a specific Excitation and Emission Spectra
wavelength and liberate energy in the form of emission of
light at a higher wavelength. The general principles can be Excitation Spectrum
illustrated by a Jablonski diagram (Papageorgiou and
Govindjee 2004; Rost 1995; Zude 2008), as shown in The excitation spectrum is defined as the relative efficiency
Fig. 1. of different wavelengths of exciting radiation in causing
The first step (1) is the excitation, where light is fluorescence. The shape of the excitation spectrum should
absorbed by the molecule, which is transferred to an be identical to that of the absorption spectrum of the
electronically excited state, meaning that an electron goes molecule and independent of the wavelengths at which
from the ground singlet states, S0, to an excited singlet fluorescence is measured. However, this is seldom the case
state, S′1. This is followed by a vibrational relaxation or because the sensitivity and the bandwidth of the spectro-
internal conversion (2), where the molecule undergoes a photometer (absorbance spectrum) and the spectrofluorim-
transition from an upper electronically excited state to a eter (excitation spectrum) are different. In addition, for
lower one, S1, without any radiation. Finally, the emission many food samples, scattering properties and energy
occurs (3), typically 10−8 s after the excitation, when the transfer between neighboring molecules could contribute
electron returns to its more stable ground state, S0, to this difference. A general rule of thumb is that the
emitting light at a wavelength according to the difference strongest (generally the longest) wavelength peak in the
in energy between the two electronic states. This expla- excitation spectrum is chosen for excitation of the sample.
nation is somewhat simplified. In molecules, each elec- This minimizes possible decomposition caused by the
tronical state has several associated vibrational states. In shorter wavelength, higher energy radiation.
the ground state, almost all molecules occupy the lowest
vibrational level. By excitation with ultraviolet or visible Emission Spectrum
light, it is possible to promote the molecule of interest to
one of several vibrational levels for the given electroni- The emission spectrum of a compound results from the
cally excited level. This implies that absorption and radiation absorbed by the molecule. The emission spectrum
Table 1 Overview of the literature survey on fluorescence of different food systems
366

Fluorophore Dairy products Meat Fish Egg Edible oils Cereal Sugar Fruit and vegetable Bacteria

Amino acids Boubellouta and Dufour Allais et al. (2004), Dufour et al. Karoui et al. Karoui et al. (2006a), Baunsgaard et al. Noh and Lu (2007), Ammor et al.
and nucleic (2008), Dufour and Dufour and Frencia (2003), Karoui (2006c, Zandomeneghi (2000a, b), Bro Seiden et al. (1996) (2004), Leblanc
acids Riaublanc (1997), (2001), Frencia et et al. (2006f) d2007d, e) (1999) (1999), Karoui and Dufour
Dufour et al. (2000), al. (2003), Lebecque et al. (2007a), (2002, 2004),
Hammami et al. (2010), et al. (2003), Møller Munck et al. Leriche et al.
Herbert et al. (1999, et al. (2003), (1998), Nørgaard (2004),
2000), Karoui and Sahar et al. (2009a) (1995), Ruoff et Tourkya
Dufour (2003, 2006), al. (2006) et al. (2009)
Karoui et al. 2004a, b,
2005a, b, c, 2006b, e,
2007c, Kulmyrzaev et
al. (2005), Liu and
Metzger (2007),
Mazerolles et al.
(2001), Rouissi et al.
(2008), Schamberger
and Labuza (2006),
Zaïdi et al. (2008)
Collagen Egelandsdal et al.
(1996, 2002, 2005),
Skjervold et al.
(2003), Swatland
(1987), Swatland and
Findlay (1997),
Swatland et al.
(1995a, b)
Chlorophyll Engelsen (1997), De Ell et al. (1996),
Guimet et al. (2004, Hagen et al. (2006),
2005), Kyriakidis Lötze et al. (2006),
and Skarkalis Moshou et al. (2005)
(2000), Poulli et al.
(2005, 2007),
Zandomeneghi et al.
(2005)
Ferulic acid Symons and Dexter
(1991, 1992, 1993,
1994)
Maillard Liu and Metzger (2007), Karoui et al. Baunsgaard et al.
products Schamberger and (2006c, d) (2000a, b)
Labuza (2006)
NADH Kulmyrzaev et al. Dufour et al. Ghosh et al. (2005) Ammor et al.
(2005) (2003), Karoui (2004), Leblanc
et al. (2006f) and Dufour
(2002, 2004),
Tourkya et
al. (2009)
Food Bioprocess Technol (2011) 4:364–386
FADH Kulmyrzaev et al. (2005)
Oxidation Karoui et al. (2007b), Gatellier et al. (2007), Hasegawa et al. Engelsen (1997),
products Wold et al. (2002, Møller et al. (2003), (1992), Olsen et Guimet et al. (2004,
2005) Olsen et al. (2005), al. (2006) 2005), Sikorska et
Sahar et al. (2009b), al. (2004, 2005)
Veberg (2006),
Veberg et al. (2006)
Polyphenols Sikorska et al. Baunsgaard et al.
(2004, 2005), (2000a, b), Karoui
Zandomeneghi et al. (2007a), Ruoff
et al. (2005) et al. (2006)
Retinol Boubellouta and Dufour Karoui et al.
(vitamin A) (2008), Dufour and (2006c, d,
Riaublanc (1997), Dufour 2007d, e)
et al. (2000), Hammami
et al. (2010), Herbert et
Food Bioprocess Technol (2011) 4:364–386

al. (1999, 2000), Karoui


and Dufour (2003, 2006),
Karoui et al. (2004a, b,
2005a, b, c, 2006b, e,
2007c), Kulmyrzaev et al.
(2005), Liu and Metzger
(2007), Mazerolles et al.
(2001), Rouissi et al.
(2008), Zaïdi et al. (2008)
Riboflavin Boubellouta and Dufour Zandomeneghi
(vitamin B2) (2008), Hammami et al. et al. (2003)
(2010), Karoui and Dufour
(2003, 2006), Karoui et al.
(2005c, 2006b, e, 2007c),
Liu and Metzger (2007),
Rouissi et al. (2008),
Zaïdi et al. (2008)
Tocopherols Guimet et al. (2004,
(vitamin E) 2005), Kyriakidis
and Skarkalis
(2000), Poulli et al.
(2005, 2007),
Sikorska et al.
(2004, 2005),
Zandomeneghi et al.
(2005)
367
368 Food Bioprocess Technol (2011) 4:364–386

is the relative intensity of radiation emitted at various sample. However, it can be beneficial and informative
wavelengths. In theory, the quantum efficiency and the to obtain the entire fluorescence landscape (also known
shape of the emission spectrum are independent of the as two-dimensional fluorescence spectroscopy) in order
wavelength of the excitation radiation. In practice, this is to find the exact excitation and emission maxima, as well
not the case. Indeed, it has been shown that fluorescence as the correct structure of the peaks. Furthermore, it
of chlorophyll from a green leaf has a lower short facilitates more appropriate analysis of fluorescence data
wavelength emission maximum when excited with green from complex samples with more fluorophores present
light than when excited with blue light (Buschmann (Christensen et al. 2006).
2007). Green light penetrates more deeply into the leaf
since it is less absorbed than blue light and the green light- Factors Affecting Fluorescence Intensity
excited fluorescence from more inside the leaf is more
readily re-absorbed by the chlorophylls on its way to the Several factors related to the nature and the concentration
sample surface. The re-absorption of fluorescence is of fluorophores of food samples influence the fluorescence
particularly high in the short wavelength fluorescence intensity.
where it overlaps with the absorption spectrum of
chlorophyll. If the exciting radiation is at wavelength that Quenching
differs from the wavelength of the absorption peak, less
radiant energy will be absorbed and hence less will be Fluorescence quenching represents any process leading to a
emitted. decrease in fluorescence intensity of the sample (Lakowicz
1983). It is related to the deactivation of the excited
Stokes Shift molecule by either intra- or intermolecular interactions.
There are two types of quenching: statistic and dynamic.
According to the Jablonski diagram (Fig. 1), the energy of The first occurs when the formation of the excited state is
emission is lower than that of excitation. This implies that inhibited due to a ground state complex formation in
the fluorescence emission occurs at higher wavelengths which the fluorophore forms non-fluorescent complexes
than the absorption (excitation). The difference between with a quencher molecule. Dynamic or collisional quench-
the excitation and emission wavelengths is known as ing refers to the process when quenchers deactivate the
Stokes shift (Valeur and Bochon 2001), as indicated with behavior of the excited state after its formation. The
the arrow in Fig. 2, marking the difference between the excited molecule will be deactivated by either intramolec-
excitation and emission spectrum of tryptophan fluores- ular interaction (collision) or intermolecular activity
cence spectra scanned on milk submitted to ultra-high (interaction with other molecules). One of the best-
temperature. known quenchers is oxygen. A higher temperature also
  results in larger amount of collisional quenching due to the
 1 1 increased velocities of molecules. Resonance energy
Stokes shift cm1 ¼ 107  ; ð2Þ
lex lem transfer can also be considered as dynamic quenching,
since an interaction between the donor and acceptor
where λ ex and λ em are the maximum wavelengths molecules could take place inducing a full or partial
(nanometer) for excitation and emission, respectively. deactivation of the excited fluorophore (donor). The
Normally, the emission spectrum for a given fluoro- energy transfer does not involve emission of light, but a
phore is a mirror image of the excitation spectrum, as dipole–dipole interaction between the donor and acceptor
seen to some extent in Fig. 2 for tryptophan. The general molecule.
symmetric nature is a result of the same transitions being
involved in both absorption and emission and the Concentration and Inner Filter Effect
similarities of the vibrational levels of S0 and S1.
However, there are several exceptions, since several The equation defining the relationship of fluorescence
absorption bands can be observed in the excitation intensity to concentration is:
spectrum but only the last peak is observed in the emission 
spectrum, representing the transition from S1 to S0. The If ¼ fI0 1  10"lc ;
fluorescence of vitamin A, as seen in Fig. 3, is an example
of this, with three absorption peaks and only one where If is the fluorescence intensity, f the quantum yield,
emission peak. Normally, only emission or excitation I 0 the intensity of the incident light, ε the molar
spectra (i.e., one excitation or emission wavelength) are absorptivity, l the optical depth of the sample, and c the
recorded when investigating the fluorescence of a molar concentration of the fluorophore.
Food Bioprocess Technol (2011) 4:364–386 369

S’1
polar environments, the fluorophore in excited state will
2
S relax to a lower energy state of S1. This means that the
emission of polar fluorophores will be shifted towards
Energy
longer wavelengths (lower energy) in more polar solvents.
The structure of macromolecules and the location in
hνex 1 hνem 3 macromolecules can also have a large effect on the
fluorescence emission and quantum yield of a fluorophore.
Temperature, pH, and color strongly affect the fluorescence
signal. Increased temperature leads to increased movement
S0 of the molecules, and thereby more collisions, thus
Fig. 1 Jablonski diagram showing the basic principle in fluorescence
inducing a reduced fluorescence signal. It is therefore
spectroscopy important that all samples in an experiment present the
same temperature. The pH value affects the fluorescence,
According to Lakowicz (1983), for low absorbance and most hydroxyl aromatic compounds fluoresce better at
(<0.05), the equation can be written as: high pH (Guilbaut 1989). The color of the sample can affect
both the shape and the intensity of the spectra. Dark
If ¼ 2:3f I0 " l c;
samples will reabsorb more of the fluorescence than bright
where If is the fluorescence intensity, f the quantum yield, samples.
I 0 the intensity of the incident light, ε the molar
absorptivity, l the optical depth of the sample, and c the Scatter
molar concentration of the fluorophore. The decrease is in a
part caused by an attenuation of the excitation beam in the Scattering of the incident light affects the fluorescence
areas of the solution in front of the detection system and by signal. As mentioned in the previous section, the absor-
the absorption of the emitted fluorescence within the bance of the sample measured plays an important role in
solution. This is defined as the inner cell or inner filter fluorescence measurements. Especially in turbid solutions
effect. The equation expressing the fluorescence intensity and solid opaque samples (like most foods), the amount of
indicates that there are three major factors other than scattered and reflected light affects the measurements
concentration that could affect the fluorescence intensity: considerably, with respect to both the sampling (i.e., the
optical depth of the sampling) and the obtained (fluores-
1. The quantum efficiency  ; the greater the  , the
cence) signal. Scattered light can be divided into Rayleigh
greater the fluorescence intensity.
and Raman scatter.
2. The intensity of incident light I0; a more intense source
Rayleigh scatter refers to the scattering of light by
will yield greater fluorescence. In actual practice, a very
particles and molecules smaller than the wavelength of the
intense source can cause photodecomposition of the
light. Rayleigh is so-called elastic scatter, meaning that no
sample. Hence, one compromise is a source of moderate
energy loss is involved, so the wavelength of the scattered
intensity (such as a mercury or xenon lamp).
light is the same as that of the incident light. Rayleigh
3. The molar absorptivity of the compound, ε. In order to
scatter can be observed as a diagonal line in fluorescence
emit radiation, a molecule must first absorb radiation.
landscapes for excitation wavelengths equaling the emis-
Hence, the higher the ε, the better the fluorescence
sion wavelength. The signal from fluorophores with little
intensity of the compound will be.
Stokes shift will be situated close to the scattering line, and
It should be remembered that the overall fluorescence therefore be most affected by Rayleigh scatter. Due to the
intensity of a given sample is expressed as the sum of the construction of grating monochromators used for excitation
fluorescence contribution from each of the inherent fluoro- in most spectrofluorometers, some light at the double
phores present in the sample. However, due to the complex wavelength of the chosen excitation will also pass through
systems of food products, the fluorescence intensity may not to the sample. For this reason, an extra band of Rayleigh
be additive because the quenching phenomenon and inter- scatter, so-called second-order Rayleigh, will typically
actions with the molecular environment of the fluorophores appear in fluorescence measurement for emission wave-
may take place. lengths at twice the given excitation wavelength. Rayleigh
scatter can be disregarded by measuring and considering
Molecular Environment the fluorescence signal only between the first- and second-
order Rayleigh scatter.
The local environment of a fluorophore has an important Raman scatter is inelastic scatter due to absorption and
effect on the shape of the fluorescence spectra. In more re-emission of light coupled with vibrational states. A
370 Food Bioprocess Technol (2011) 4:364–386

Fig. 2 Excitation (full line) and 0.16


emission (dotted line) trypto-
phan fluorescence spectra
recorded on UHT milk

Fluorescence intensity (a.u.)


0.12

0.08

0.04

0
245 265 285 305 325 345 365 385
Wavelength (nm)

constant energy loss will appear for Raman scatter, meaning lengths; a detector, which converts the emitted light to an
that the scattered light will have a higher wavelength than electric signal; and a unit for data acquisition and analysis.
that of the excitation light, with a constant difference in The sampling geometry can have a substantial effect on the
wavelength. In liquid samples, the solvent is decisive in the obtained fluorescence signal. If absorbance is less than 0.1,
amount and nature of Raman scatter, while for solid the intensity of the emitted light is proportional to the
samples it will typically be an expression of the bulk fluorophore concentration and excitation and emission
substances. Raman scatter can in most cases be neglected spectra are accurately recorded by a classical right-angle
because of its weak contribution to the fluorescence signal. fluorescence device. In this case, the excitation light travels
into the sample from one side, and the detector is
positioned at right angles to the center of the sample.
Instrumentation When the absorbance of the sample exceeds 0.1, the
intensity of emission and excitation spectra decreases and
The basic setup for an instrument for measuring steady- excitation spectra are distorted. To avoid these problems,
state fluorescence is shown in Fig. 4. The spectrofluorim- dilution of samples (when it is possible, i.e., liquid samples)
eter consists of a light source (generally xenon or mercury is currently performed so that their total absorbance will be
lamp); a monochromator and/or filter(s) for selecting the less than 0.1. However, the results obtained on diluted
excitation wavelengths; a sample compartment; a mono- solutions of food samples cannot be extrapolated to native
chromator and/or filter(s) for selecting the emission wave- concentrated samples since the organization of the food

Fig. 3 Excitation (full line) and 0.16


emission (dotted line) vitamin A
fluorescence spectra recorded on
Fluorescence intensity (a.u.)

UHT milk
0.12

0.08

0.04

0
250 300 350 400 450 500
Wavelength (nm)
Food Bioprocess Technol (2011) 4:364–386 371

Fig. 4 Basic setup of a Light Emission


spectrofluorimeter
Monochromator Photodetector
Interference filter

Excitation Sample holder Data acquisition and analysis

Monochromator
Interference filter

Right-angle Front-face

matrix is lost. In addition, the dilution may change the regression (PLS), factorial discriminant analysis (FDA),
concentration of other relevant fluorescent species below or parallel factor analysis (PARAFAC), etc. have proven to be
close to the detection limit of fluorescence. Moreover, for powerful methods for the extraction of valuable information
solid samples, the dilution cannot be realized (e.g., meat, (Boubellouta and Dufour 2010; Christensen et al. 2006;
cheese). To avoid these problems, FFFS can be used Hammami et al. 2010; Kulmyrzaev and Dufour 2010).
(Fig. 4). In this manner, it is possible to measure more turbid
or opaque samples, since the signal becomes more indepen-
dent of the penetration of the light through the sample. Applications of Fluorescence in Foods and Drinks
However, when front-face sampling is used, the amount of
scattered light detected will increase due to the higher level of Recently, the application of fluorescence spectroscopy in
reflection from the surface topology of the sample and sample combination with multidimensional statistical techniques
holder. To minimize these effects, it is recommended that the for the evaluation of food quality has increased. In most of
sample is not placed with its surface oriented at an angle of 45° the research papers, the obtained fluorescence signal was
to the incident beam, but rather at 30°/60° to the light source assigned to specific fluorophores after fixing the excitation
and the detector (Lakowicz 1983). or the emission wavelength.

Dairy Products
Data Analysis
Dairy products contain several intrinsic fluorophores, which
Data analysis of fluorescence spectra has been well represent the most important area of fluorescence spectrosco-
established by Smilde et al. (2004). Fluorescence is py. They include the aromatic amino acids and nucleic acids
inherently multidimensional. Indeed, multidimensional (AAA+NA) tryptophan, tyrosine, and phenylalanine in
fluorescence signals recorded from a sample can conve- proteins; vitamins A and B2; nicotinamide adenine dinucle-
niently be presented as a matrix of fluorescence intensities otide (NADH) and chlorophyll; and numerous other com-
as a function of excitation and/or emission wavelengths. pounds that can be found at a low or very low concentration
Due to the neighboring wavelengths, highly correlated data in food products.
present in emission and excitation spectra have been Dufour and Riaublanc (1997) investigated the potential
pointed out (Smilde et al. 2004). In this case, principal of FFFS to discriminate between raw, heated (70 °C for
component analysis (PCA), common component and 20 min), homogenized, and homogenized and heated milks.
specific weights analysis (CCSWA), partial least squares The authors applied PCA to the tryptophan and vitamin A
372 Food Bioprocess Technol (2011) 4:364–386

fluorescence spectra, and good differentiation between milk By applying FDA, Feinberg et al. (2006) found that
samples as a function of homogenization and heat treatment tryptophan fluorescence spectra could be considered well-
applied to milk samples was observed. They concluded that adapted to discriminate sterilized milks and probably
the treatments applied to milk induced specific modifica- pasteurized milks from the other milk samples. However,
tions in the shape of the fluorescence spectra. Recently, this intrinsic probe failed to discriminate the other types of
Kulmyrzaev et al. (2005) confirmed these earlier findings. milk. An explanation could be that fluorescence spectra were
In their research, the emission and excitation spectra of recorded in the pH 4.6 soluble fraction of the milk sample,
different intrinsic probes (i.e., AAA+NA, NADH, and inducing a loss of information contained in milk samples.
FADH) were used to evaluate changes in milk following Regarding milk coagulation, Herbert (1999) and Herbert
thermal treatments in the range of 57–72 °C for 0.5– et al. (1999) used FFFS to monitor milk coagulation at the
30 min. The PCA applied to the normalized spectra allowed molecular level. Three different coagulation processes have
good discrimination of milk samples subjected to different been studied: the glucono-δ-lactone (GDL), the rennet-
temperatures and times; the obtained results were con- induced coagulation system, and a mixed GDL and rennet-
firmed recently by Boubellouta and Dufour (2008) who induced coagulation system. Emission fluorescence spectra
discriminated milk samples according to heating in 4–50 °C of the tryptophan were recorded for each system during the
and acidification (pH ranging from 6.8 to 5.1). Boubellouta milk coagulation kinetics. By applying the PCA to normal-
et al. (2009) determined the effect of the adding calcium, ized fluorescence spectra data sets of the three systems,
phosphate, and citrate at different levels (i.e., 3, 6, and detection of structural changes in casein micelles during
9 mM) on the molecular structure of skimmed milks; the coagulation and discrimination of different dynamics of the
addition of phosphate induced molecular change (observed three coagulation systems was achieved. Herbert et al. (1999)
by mid-infrared and synchronous fluorescence) that was concluded that FFFS allows the investigation of network
different than that of calcium and citrate; the origin of this structure and molecular interactions during milk coagulation.
difference was not depicted by the authors. In these three Most of the aforementioned studies regarding discrimi-
latter research studies, milk samples were heated at only nation of milk were performed at a laboratory scale on
relatively low temperatures allowing the non-monitoring of extreme and controlled samples. Milk products from
the development of Maillard browning reaction; this was mountain areas are reputed to have specific organoleptic
realized after by Schamberger and Labuza (2006); the and nutritional qualities (Bosset et al. 1999; Coulon and
fluorescence spectra of milks were processed for 5, 15, 20, Priolo 2002; Renou et al. 2004), and the tracing of milk
25, and 30 s in 5 °C increments from 110 to 140 °C and production sites is therefore important in order to avoid
found to be well correlated with hydroxylmethylfurfural. fraud. In this context, the potential of FFFS to discriminate
Indeed, the R2 values of 0.95 were found continuously between milks according to their geographical origin was
throughout the emission wavelength range of 394 to explored. Forty milk samples—8 produced in lowland areas
447 nm. In addition, the fluorescence levels increased with (430–480 m), 16 produced in mid-level areas (720–860 m),
higher time–temperature combinations. One of the main and 16 produced in mountain (1,070–1,150 m) areas—from
conclusions of this study was that FFFS could be considered a the Haute-Loire department in France at key periods of
promising method for measuring Maillard browning in milk; animals feeding were analyzed (Karoui et al. 2005c).
the authors encouraged the use of this technique for on-line Tryptophan fluorescence spectra, AAA+NA spectra, and
monitoring of thermal processing of milk. Later, Liu and riboflavin spectra were recorded directly on milks, with
Metzger (2007) confirmed the aforementioned results follow- excitation wavelengths set at 290, 250, and 380 nm,
ing the use of FFFS for monitoring changes in non-fat dry respectively. The excitation spectra of vitamin A were also
milk (n=9) collected from three different manufacturers and recorded, with the emission wavelength set at 410 nm. By
stored at four different temperatures (4, 22, 35, and 50 °C) applying FDA to the spectral collection, a trend to a good
for 8 weeks. Different intrinsic probes (fluorescent Maillard separation between milks as a function of their origins was
reaction products (FMRP), riboflavin, tryptophan, and observed. The best results were obtained with AAA+NA
vitamin A) were used, and each of the considered spectral fluorescence spectra, since 81.5% and 76.9% of the
data sets allowed good discrimination of milk samples kept calibration and validation spectra, respectively, were cor-
at 50 °C from the others. In addition, good discrimination of rectly classified. However, some misclassification occurred
milk samples as a function of the storage time was observed. between milks produced in mid-level areas and the other
In a similar approach, Feinberg et al. (2006) used fluores- milk samples. In the same context, FFFS has demonstrated
cence spectroscopy to identify five types of heat treatments its potential for monitoring ewe’s milk samples according
(pasteurization, high pasteurization, direct ultra-high temper- to the feeding system, i.e., scotch bean versus soybean
ature (UHT), indirect UHT, and sterilization) of 200 meals (Hammami et al. 2010; Rouissi et al. 2008) and
commercial milk samples stored at 25 and 35 °C for 90 days. genotype (Zaïdi et al. 2008).
Food Bioprocess Technol (2011) 4:364–386 373

Regarding the use of FFFS for monitoring the quality of explored the potential of FFFS to discriminate between
cheeses during ripening, Dufour et al. (2000) and Mazerolles different soft cheese varieties. Tryptophan and vitamin A
et al. (2001) used FFFS to monitor 16 semi-hard cheeses spectra were acquired on the cheese samples. The environ-
produced and ripened under a controlled scale. By applying ment of the tryptophan residues was found to be relatively
PCA to the normalized tryptophan fluorescence spectra, more hydrophilic for the ripened cheeses than for those at
good discrimination of cheeses presenting a ripening time of the young stage. This phenomenon was attributed to partial
21, 51, and 81 days was observed, while an overlap was proteolysis of caseins during ripening, resulting in an
observed between cheeses aged 1 day and those aged increase of tryptophan exposure to the solvent. To test the
21 days. The spectral pattern of tryptophan indicated a red accuracy of FFFS in differentiating between the eight soft
shift of aged cheeses suggesting that the environment of cheeses, the authors applied FDA to the most relevant PCs,
ripened cheeses was more hydrophilic than that of young (1- and good discrimination of cheeses was observed, with
day-old) cheeses. This phenomenon was assigned to partial better results obtained with vitamin A spectra (96% and
proteolysis of casein as well as to the salting phenomenon, 93% for the calibration and validation samples, respective-
which may induce some changes in the tertiary and ly) than with tryptophan spectra (95% and 92% for the
quaternary structures of casein micelles. Regarding the calibration and validation samples, respectively). However,
fluorescence spectra of vitamin A, two shoulders located at in their investigations, samples were studied at the center of
295 and 305 nm and a maximum located at 322 nm were the cheese, which could induce some misclassification
observed (Dufour et al. 2000). In addition, the shape of the between the investigated cheeses. In the case of soft
spectra changed with ripening time. By applying PCA to the cheeses, protein breakdown, lipolysis, pH, etc. differ
normalized vitamin A spectra, better discrimination of significantly between the surface and the center. To reply
cheeses aged 21, 51, and 81 days from those aged 1 day with this request, the matrix structure of three retailed soft
was achieved. The authors determined the link between the cheeses, produced each with different manufacturing
data recorded by mid-infrared (MIR) and fluorescence by process, was studied from the surface to the center of the
using canonical correlation analysis (CCA). Correlation cheese using FFFS, among other techniques (Karoui and
coefficients of 0.58 or more were obtained suggesting that Dufour 2003). Cheese slices, 5-mm thick, were cut from the
fluorescence and MIR spectra might provide a common surface to the center of the samples. PCA applied to the
description of the investigated cheese samples during tryptophan fluorescence spectra recorded for each cheese
ripening. variety showed good discrimination of cheese samples as a
Karoui et al. (2006b) continued this work by recording function of their location. The environment of tryptophan
tryptophan, vitamin A, and riboflavin spectra of 12 semi- residues was found to be more heterogeneous in the surface
hard cheeses (Raclette) of 4 different brands, which were samples than in the center samples; this was attributed to
produced during summer period at the industrial level. By the changes in the extent and type of protein–protein
applying CCSWA to the spectral data sets and physico- interactions in the protein network depending on the
chemical data, good discrimination of the four brands was sampling zone. One of the limiting points of this study
observed. The same research group (Karoui and Dufour was the low number of cheeses. In addition, only
2006) evaluated the potential of FFFS to predict the tryptophan and vitamin A fluorescence spectra were studied
rheological parameters of 20 semi-hard cheeses at the end in this research. To reply with this request, later, Karoui et
of their ripening stage (60 days) from fluorescence spectra al. (2006e, 2007c) used FFFS to investigate changes at the
recorded at a young stage (2 days old). By using tryptophan molecular level of both the external (E) and central (C)
fluorescence spectra scanned on cheeses aged 2 days and at zones of 15 ripened soft cheeses produced according to
20 °C, the storage modulus (G′), loss modulus (G″), strain, traditional and stabilized cheese-making procedures. The
tan (δ), and complex viscosity (η*) were predicted by using CCSWA was applied to the tryptophan, vitamin A, and
PLS regression with leverage correction with R higher than riboflavin spectral data sets, and the plane defined by the
0.97. The obtained results were confirmed recently by common components 1 (q1) and 3 (q3) showed clear
Boubellouta and Dufour (2010), reporting that synchronous discrimination between the cheese varieties and sampling
fluorescence spectroscopy could be used for the determi- zones. From the obtained results, it was concluded that
nation of fat melting and cheese melting of two cheese CCSWA allowed very efficient management of all the
varieties (i.e., Comté and Raclette). spectroscopic information collected on the investigated soft
In another context, FFFS has been used for the cheeses. Each of the fluorophore provides information,
authentication of different varieties of soft, semi-hard, and which can be used for recognizing the cheese variety and
hard cheeses during ripening at the retail stage (Herbert sampling zone. The CCSWA method sums up this
1999; Herbert et al. 2000; Karoui and Dufour 2003; Karoui information using two common components (q1 and q3),
et al. 2003, 2004a, b, 2005a, b). Herbert et al. (2000) taking into account the relation between the different
374 Food Bioprocess Technol (2011) 4:364–386

fluorescence data sets. The result obtained from CCSWA tion that takes place during ripening seemed to present
did not observed with the PCA performed separately on lesser effect than did light and oxygen.
each of fluorescence spectra, illustrating that CCSWA
methodology allowed efficiently the use of all the spectro- Meat and Meat Products
scopic information provided by the three intrinsic probes. In
another study, Karoui et al. (2005a) attempted to discrim- Research regarding the application of FFFS for the evalua-
inate 25 Gruyère and L’Etivaz Protected Designation of tion of meat products has focused on the measurements of
Origin cheeses. Emission spectra were scanned following fluorescence from collagen, adipose tissues, and protein
excitation at 250 and 290 nm, and excitation spectra following (Newman 1984; Jensen et al. 1989; Frencia et al. 2003).
emission at 410 nm. By applying FDA, 100% correct The collagen in connective tissue is known to be an
classification was obtained from the emission and excitation important parameter of meat quality, as it is related to the
spectra, suggesting the use of FFFS as an accurate technique tenderness and texture of the meat. Collagen exists in several
for the determination of the geographic origin of cheeses. different genetic forms, four of which have been found to be
These findings were fully supported on Emmental cheeses present in muscle: types I, III, IV, and V. Types I, III, and IV
originating from different European countries and manufac- presented similar fluorescent characteristics when they were
tured during both winter and summer seasons (Karoui et al. excited in the 330–380-nm spectral region (Hildrum et al.
2004a, b, 2005b). One of the strong conclusions of these 2006). Elastin, another important fluorophore in meat,
studies was that FFFS allows a good discrimination between presents quite similar fluorescence properties to those of
Emmental cheeses produced from raw milk and those made collagen types I, III, and IV (Egelandsdal et al. 2005).
with thermized milk. This was supported by the accuracy of Adipose tissue contains fluorescent molecules that are
FFFS to predict chemical parameters collected from the E specific for fat. Indeed, it has been shown by several
and C zones of 15 soft cheeses being assessed (Karoui et al. authors (see, for example, Ramanujam 2000; Skjervold et
2006e). The PLS regression applied to the normalized al. 2003) that the fat-soluble vitamins A, D, and K exhibit
vitamin A fluorescence spectra provided the highest values fluorescence in the 387–480-nm spectral region after
of R2, since values of 0.88, 0.86, 0.86, and 0.84 were found excitation at 308–340 nm. Swatland (Swatland 1987;
for fat, dry matter, fat in dry matter, and water-soluble Swatland et al. 1995a, b; Swatland and Findlay 1997)
nitrogen, respectively. could be considered the pioneer in the field of meat, with a
Finally, FFFS was used for monitoring the oxidation of series of papers on different aspects of the use of FFFS,
dairy products. Good estimation of the sensory analyses starting in 1987. His work focused on measuring collagen
from FFF spectra of both sour cream (Wold et al. 2002) and and elastin fluorescence from the connective tissues in
cheese (Wold et al. 2005) has been achieved. Wold et al. meat. The author reported that after excitation at 365 nm,
(2005) showed that naturally occurring porphyrin and the fluorescence emission spectra of adipose tissue
chlorophylls play an important role as photosensitizers in exhibited a maximum at 510 nm with a secondary plateau
dairy products. The degradation of these components varying from 430 to 450 nm (Swatland 1987). The obtained
showed higher correlation with sensory measured lipid fluorescence spectra of various meats were found to be
oxidation. Recently, the feasibility of the use of FFFS as a correlated with biochemical and sensory analyses such as
non-destructive technique for monitoring oxidation at the chewiness (Swatland et al. 1995a), palatability (Swatland et
molecular level of semi-hard cheeses, made with cow’s al. 1995b), and toughness (Swatland and Findlay 1997).
milk and collected during both the grazing period (summer) Most of the studies realized by Swatland and co-workers
and the stabling period (autumn), was examined at the were analyzed using univariate data analytical approach, by
surface (20 mm from the rind) and the inner (40 mm from the comparison of single wavelength or extracted fluores-
the rind) layers throughout the ripening stage—i.e., 2, 30, cence peak features. Although interesting results were
and 60 days old by Karoui et al. (2007b). By applying FDA achieved, the use of multivariate statistical analyses is
to the 400–640-nm emission fluorescence spectra recorded needed for curve resolution and useful quantitative meas-
at the surface layer, correct classification was observed for urements due to the complexity of fluorescence spectra.
100% and 91.7% for the calibration and the validation Egelandsdal et al. (1996) applied PCA and PLS regression
spectra, respectively. With regard to the samples cut from to the fluorescence spectra scanned on meat products after
the inner layers, the authors stated that the 400–640-nm excitation at between 300 and 400 nm for studying isolated
emission fluorescence spectra failed to discriminate cheeses perimysial sheets from a type I muscle and found a high
that were either 2 or 30 days old. The main conclusion of correlation between perimysial breaking strength and
this study was that throughout ripening the riboflavin fluorescence emission spectra recorded after excitation at
component was affected primarily by oxygen and light 335 nm. Wold et al. (1999a, b) later confirmed these
(Marsh et al. 1994), while the physicochemical modifica- previous investigations and suggested that it would be
Food Bioprocess Technol (2011) 4:364–386 375

possible to measure the amount of connective tissue in muscles (Hildrum et al. 2006). Recently, tryptophan fluo-
ground meat by using an excitation wavelength of 380 nm. rescence spectra scanned on two beef muscles (longissimus
They have also showed that both connective tissue and thoracis and infraspinatus) 2 and 14 days post mortem
intramuscular fat content could be measured using an showed a maximum located around 336 nm (Dufour and
excitation wavelength of 332 nm. Egelandsdal et al. Frencia 2001). In addition, the maximum emission of aged
(2002) studied six different batches of beef longissimus muscles showed a shift to higher wavelengths (red shift).
dorsi samples originating from 151 animals by using FFFS The PCA performed on spectra showed good discrimination
and Warner–Bratzler (WB) peak values. By applying PLS of samples according to the muscle type and aging.
regression, poor to good (R=0.45–0.84) correlations be- However, with only a limited number of meat samples, the
tween WB peak values and the emission spectra were models suffered from over-fitting and consequently were not
obtained. In addition, minor difference in predictability was very robust against the inclusion or exclusion of samples.
observed using excitation wavelengths at 332 or 380 nm. Further analyses with more samples are necessary to
The emission wavelengths containing the most relevant substantiate these models. This would allow more variability
information about WB peak values were found in the 360– of the chemical properties and thus development of general
500-nm spectral range. Emission wavelengths around mathematical models for better accuracy of the FFFS
375 nm, following excitation at 332 nm, were found to be technique. Frencia et al. (2003) therefore assessed the
related to a component in the perimysial tissue, most likely potential of FFFS to discriminate between five muscle types
present in collagen I or III. In another study, FFFS was presenting different levels of collagen contents at two points
explored to predict the age of Parma hams produced from in time (2 and 14 days post mortem). Applying FDA to the
two muscles (semi-membranosus and biceps femoris) and tryptophan fluorescence spectra, correct classification rate of
aged 3 months (young), 11 and 12 months (matured), and 82% was obtained. The authors concluded that FFFS is a
15 and 18 months (aged) by Møller et al. (2003). Using powerful technique that allows a relatively good identifica-
PLS regression, prediction of the age was considered as tion of muscle types according to maturation. Preliminary
good, with a relative error of prediction of approximately results have shown that results obtained at the molecular
1 month; also, a good correlation between fluorescence and level by FFFS are related to the macroscopic levels (sensory
chemical, sensory, and physical parameters was found. and rheology data sets). Indeed, by applying CCA to the
Egelandsdal et al. (2005) tried to throw more light on the fluorescence spectra (spectrofluorimeter with a front-face
phenomena affecting the fluorescence signal and the ability device or coupled to a fiber-optic) and mechanical properties
of the FFFS technique to quantify collagen contents. Beef (texturometer) or sensory attributes, a strong correlation
masseter and latissimus dorsi and pork glutens medius between the different methods was found. The authors
muscles, among others, were chosen for their wide differ- concluded that a common description of the samples was
ences in color and connective tissue quality and content. possible from both the fluorescence and the rheology or
PLS regression was applied to their fluorescence spectra in sensory data, which was later confirmed by the investigation
order to predict collagen (measured as hydroxyproline), and of Lebecque et al. (2003) on 25 longissimus thoracis samples
good results were obtained (root mean square error of from animals presenting different ages (between 2.5 and
prediction (RMSEP) of 0.55%). Similar prediction results 8 years) and sexes. The authors depicted that the phenomena
were obtained with complex sausage batters consisting of observed at the molecular and macroscopic levels were
different kinds of muscles and presenting a large span in related to the changes in the texture of meat during aging.
myoglobin and realistic ranges in collagen and fat. One of The obtained results were confirmed by Allais et al. (2004)
the interesting results obtained in this study was that FFFS on meat emulsion and frankfurter reporting that high
gave lower prediction errors for collagen content than did correlation was obtained from fluorescence spectra and
near-infrared (NIR) reflectance when applied to the same rheology methods.
batters. Recently, Sahar et al. (2009a) tried to predict some Lipid oxidation is one of the factors limiting the quality
parameters in meat with excitation set at 290 nm (emission and acceptability of meat and meat products (Veberg 2006).
305–400 nm) and the obtained results were not successful As explained above, lipid oxidation can be determined by
since only 53% and 55% of correct classification for protein several methods, such as 2-thiobarbituric acid, sensory
and cooking loss, respectively, in the validation data sets analysis, and dynamic headspace gas chromatography
were obtained. combined with mass spectrometry. Veberg et al. (2006)
Differences in the level of collagen within a muscle or used FFFS and other destructive methods to determine the
between different muscles led to a huge difference in level of lipid oxidation and explored the usefulness of this
tenderness (Light et al. 1985). Using excitation wave- technique for detection of low levels of lipid oxidation in
lengths set between 332 and 380 nm, FFFS found a turkey meat stored during 9 months at −10 and −20 °C. By
promising technique for estimating tenderness in such applying PLS regression between fluorescence spectra and the
376 Food Bioprocess Technol (2011) 4:364–386

values obtained by the other traditional techniques, good freeze-dried fish. Two excitation wavelengths, i.e., 370 and
correlations between fluorescence spectra and thiobarbituric 450 nm exhibiting maximum emissions at 460 and 500 nm,
acid reactive substances (TBARS), hexanal, 1-penten-3-ol, respectively, were used. For fish samples stored at 25 °C in
total components and sensory measured rancidity and the dark, an increase in the fluorescence intensity at 500 nm
intensity were found. One of the interesting conclusions was noted, while that at 460 nm remained unchanged. The
of this study was that FFFS could be considered as a fluorophores observed at 460 nm have been attributed to
sensitive and non-destructive method for early lipid the reaction between reducing sugars (e.g., glucose and
oxidation determination in turkey meat. Sahar et al. ribose) and amino acid compounds inducing activation of
(2009b) confirmed the obtained results recently on the Maillard reaction, while that observed at 500 nm has
chicken meat since FFFS was found to be able to been attributed to the lipid oxidation products. In a similar
determine heterocyclic aromatic amines in grilled meat. approach, Olsen et al. (2006) used an excitation wavelength
The obtained results confirmed the previous findings of of 382 nm to record spectra in the 450–750-nm region on
Olsen et al. (2005) who monitored poultry meat freeze- four different batches of salmon pâté stored at 4 °C for 4, 8,
stored in air at −20 °C for 26 weeks and stated that FFFS and 13 weeks. Citric acid or calcium disodium ethylene-
and gas chromatography coupled with mass spectrometry diamine tetraacetate was added as metal chelators to two
could detect oxidative changes in pork back fat earlier batches, whereas no chelator was added to the third batch.
than the sensory panel and the electronic nose could. The three investigated batches contained oil, while a fourth
However, the correlation of fluorescence spectra with one was made with the same amounts of ingredients but
sensory analyses was found to be poorer and less than that without any oil. The obtained results showed an increase in
observed between sensory analysis and gas chromatogra- the fluorescence intensity with increased storage time for all
phy with mass spectrometry. In a similar approach, the batches. In addition, the shape of the spectra changed
Gatellier et al. (2007) monitored lipid oxidation of chicken largely between samples containing oil and those without
meat by using FFFS and TBARS over 9 days. In their oil. Indeed, samples containing oil exhibited the highest
research study, three chicken genotypes representative of intensity in the range of 470–475 nm, while those with no
French production were compared (i.e., Standard, Certi- added oil presented maxima between 440 and 450 nm. By
fied, and Label). The samples were stored in darkness at applying PCA to the collection of spectral data, a clear
4 °C, and a good correlation between emission spectra difference between samples according to the storage time
recorded after excitation at 380 nm and TBARS was was observed; the largest variation in the data sets was
found. In addition, the genotype meat was found to attributed to whether the sample contained oil or not; the
present an effect on the shape of fluorescence emission storage time was the second most important factor that led
spectra, since the fluorescence intensity of the emission to this discrimination. The authors applied PLS regression
spectra of Certified and Label animals after 7 days of to estimate the age of salmon pâté. The correlation
refrigerated storage was significantly higher than that of coefficients for the sensory attributes, dynamic head-space
Standard chicken meat samples. The obtained results data, fluorescence spectra, and electronic nose sensor
confirmed previous findings of Munck (2001) reporting responses were 0.64, 0.94, 0.93, and 0.70, respectively.
that fluorescence could be used in slaughtering and cutting The corresponding RMSEP were 3.8, 1.7, 1.8, and 3.5,
by robots. respectively, illustrating that FFFS could be a suitable
technique to measure lipid oxidation. This observation is
Fish confirmed by the highest level of correlation found between
fluorescence spectra and sensory attributes, among the
The lipid fraction of marine fish has been shown to contain other analytical techniques. Recently, FFFS has been used
a high level of polyunsaturated fatty acids. During storage to monitor fish freshness for four different species—cod,
and/or processing, the degradation of polyunsaturated fatty mackerel, salmon, and whiting fillets—at 1, 5, 8, and
acids can lead to the development of primary and secondary 13 days of storage (Dufour et al. 2003). Emission spectra of
products, resulting in the formation of fluorescent com- AAA+NA, tryptophan, and NADH were scanned after
pounds and the loss of essential nutriments. Indeed, excitation at 250, 290, and 336 nm, respectively. The spectra
Gardner (1979) has shown that relatively higher fluores- of the first two excitation wavelengths showed maxima located
cence intensity was observed at longer wavelength maxima at 338 and 336 nm, respectively, while the last excitation
as the quality of fish decreased. wavelength showed two maxima located at 414 and 438 nm.
The 493/463- and 327/415-nm ratios have proved to be a For all three excitation wavelengths, the shape of the spectra
more effective index of fish quality than other common illustrated some differences according to storage time, suggest-
assessment methods. Hasegawa et al. (1992) used FFFS for ing that a fluorescence spectrum may be considered as a
the quantitative assessment of oxidative deterioration in fingerprint. Applying FDA to the tryptophan fluorescence
Food Bioprocess Technol (2011) 4:364–386 377

spectra allowed 56% of samples to be correctly classified. freshness because consumers may perceive variability in
Better classification was obtained from AAA+NA and NADH, freshness as lack of quality.
since 92% and 74% correct classification was observed, The changes that occur in eggs during storage are many
respectively. The authors concluded that AAA+NA fluores- and complex and affect the functional properties of egg
cence spectra could be considered as fingerprints that may yolk and egg albumen. These changes include thinning of
allow discrimination between fresh and aged fish fillets. albumen, increase of pH, weakening and stretching of the
Aubourg et al. (1998) used fluorescence spectroscopy to vitelline membrane, and increase in the water content of the
monitor changes that occurs in sardines stored at −18 and yolk. Freshness can be explained to some extent by
−10 °C. Sardines stored at −18 °C were sampled after 0.5, objective sensory, (bio)chemical, microbial, and physical
2, 4, 8, 12, and 24 months, and those stored at −10 °C were parameters and can therefore be defined as an objective
sampled at 3, 10, 25, 60, and 120 days. Fluorescence was attribute. Knowledge of the various descriptors of proper-
measured at two excitation–emission maxima, 327/463 and ties that are encountered in eggs immediately after laying
393/463 nm. The authors used the fluorescence ratio, must be known, as well as the changes in properties that
defined as the fluorescence intensity at 327/463 nm over take place over time. This information can be gained by
the fluorescence intensity at 393/463 nm, determined in performing controlled storage experiments that extend from
aqueous and organic solutions (phases resulted from lipid the time after laying; loss in freshness and spoilage can thus
extraction). This ratio was found to increase throughout the be monitored. Posudin (1998) assessed the potential of
whole storage time at the two temperatures when it was FFFS to determine egg freshness by using ultraviolet
determined in the aqueous solution. One of the most radiation for the quality evaluation of eggs with differing
limiting points of this study was that the use of only levels of pigmentation. The emission spectra of different
maxima of emission and excitation wavelengths could eggs showed two maxima located at 635 and 672 nm
induce some loss of information contained in the fluores- (ascribed to the pigments of porphyrin and porphyrin
cence spectra. Recently, Karoui et al. (2006f) explored the derivatives of florin and oxoflorin) after excitation at 405,
potentiality of FFFS to differentiate between fresh and frozen- 510, 540, and 557 nm. The intensity at 672 nm depends on
thawed fish. A total of 24 fish (12 fresh and 12 frozen-thawed) the egg freshness. The autofluorescence of fresh egg is
were analyzed by using excitation wavelengths set at 290 stronger than that of old one, since the intensity of
(tryptophan) and 340 nm (NADH). The emission spectra of autofluorescence depends on the amount of porphyrin on
NADH of fresh fish showed a maximum at 455 nm and a the shell surface. From these preliminary results, the author
shoulder at 403 nm, while frozen-thawed fish was character- concluded that fluorescence spectroscopy could be a
ized by a maximum located at 379 nm and a shoulder at promising approach for quantitative estimation of porphyrin
455 nm. By applying PCA to NADH spectra, good in eggs and thus determine egg freshness. Recently, FFFS
discrimination between fresh and frozen fish samples was was used to monitor egg freshness during storage (Karoui
observed, which was confirmed by applying FDA to the first et al. 2006c, d). The authors found that FMRP (excitation
five PCs of the PCA performed on the NADH spectra. Indeed, 360 nm; emission 380–580 nm) recorded on thick and thin
100% correct classification was obtained for the calibration albumens and vitamin A scanned on egg yolk (emission
and validation data sets, respectively. One of the interesting 410 nm; excitation 270–350 nm) could be considered as
conclusions of this research was that NADH fluorescence powerful tools for the evaluation of egg freshness stored at
spectra may be considered a promising tool for differentiating room temperature, while tryptophan fluorescence spectra
between fresh and frozen-thawed fish samples. recorded on thick and thin albumens and egg yolk failed to
discriminate between fresh and aged eggs. Using excitation
Eggs and Egg Products at 360 nm, the emission spectra recorded on fresh thick egg
albumen exhibited two maxima located at 410 and 440 nm,
In order to ensure high and consistent egg quality, an respectively. Similar results were obtained on thin albumen
attractive and alternative strategy for determining the state of of fresh eggs. The very characteristic fluorescence spectra of
egg freshness can be achieved by sensor technologies. These thick and thin albumen of eggs stored for a long time (i.e.,
techniques (such as NIR, MIR, fluorescence spectroscopies, 18 days or more) at room temperature showed a shoulder
etc.) appear to be promising tools for non-destructively located at 414 nm and a maximum at approximately 438 nm.
determining egg freshness. Such methods cannot eliminate In addition, as the spectra showed large differences between
the need for more detailed physicochemical analyses, but fresh thin/thick egg albumens and those stored for a long
they may help to screen for samples that require further time (29 days), the authors considered the spectra as
examination. Freshness makes a major contribution to the fingerprints for freshness identification. Indeed, thick and
quality of eggs and egg products. One of the main concerns thin albumens of fresh eggs within 2–3 days of laying had the
of the egg industry is the systematic determination of egg highest intensity at 410 nm, while aged eggs had the lowest
378 Food Bioprocess Technol (2011) 4:364–386

one. The authors concluded that the shape of FMRP is Edible Oils
correlated with storage time: thick albumen of fresh eggs
had the lowest ratio of fluorescence intensity FI440 nm/ Olive oil is an economically important product of Mediter-
FI410 nm (i.e., 1.0), while that of eggs stored for 29 days ranean countries. It has a fine aroma and pleasant taste, with
had the highest (i.e., 1.30). The changes in the FI440 nm/ excellent health benefits. The quality of olive oil ranges
FI410 nm ratio has been ascribed to the change in the from the high-quality extra-virgin olive oil (EVOO) to the
viscosity of both thick and thin egg albumens and the low-quality olive-pomace oil. EVOO is obtained from the
formation of furosine during storage (Birlouez-Aragon et fruit of the olive tree by mechanical pressing and without
al. 1998; Kulmyrzaev and Dufour 2002). Indeed, it has refining processes. Owing to its high quality, it is the most
been reported that during egg storage, a decrease in the expensive type of olive oil. For this reason, it may be
viscosity of thick albumen was observed (Lucisano et al. mislabeled or adulterated for economic reasons. Mislabel-
1996). This phenomenon has been attributed to the ing often involves false information regarding the geo-
separation of the β-fraction of ovomucin, rich in carbo- graphic origin or oil variety (Aparicio et al. 1997).
hydrate, and from the ovomucin–lysozyme complex. Adulteration involves the addition of cheaper oils; the most
However, in the study by Karoui et al. (2006c, d), eggs common adulterants found in virgin olive oil are refined
were stored at room temperature, and little attention has olive oil, residue oil, synthetic olive oil–glycerol products,
been given to the influence of temperature and relative seed oils, and nut oils (Baeten et al. 1996; Downey et al.
humidity variations on fluorescence measurements, al- 2002; Sayago et al. 2004). Owing to the low price of olive-
though Stadelman et al. (1954) observed a linear decrease pomace oil, it is sometimes used to adulterate EVOO. For
of −1.15 Haugh Units per 10 °C increase in testing this reason, a rapid method to detect such a practice is
temperature. Therefore, Karoui and co-worker have important for quality control and labeling purposes. In this
continued this work by investigating changes at the context, Zandomeneghi et al. (2005) recorded fluorescence
molecular level of 126 eggs stored at 12.2 °C and 87% spectra of EVOO using right angle and FFFS. The former
RH for 1, 6, 8, 12, 15, 20, 22, 26, 29, 33, 40, 47, and method showed considerable artifacts and deformation,
55 days (Karoui et al. 2007d). Of the intrinsic fluoro- while the latter provided spectra that are much less affected
phores tested, only PCA applied to the vitamin A by self-absorption. The authors attributed this to the self-
fluorescence spectra allowed good identification of eggs absorption phenomena when using right-angle fluores-
as a function of their storage time. By applying FDA to cence, even when the spectra are corrected for inner filter
the AAA+NA spectra, correct classification rates of 69.4% effects. In another study, Sayago et al. (2004) applied
and 63.9% were observed for the calibration and valida- fluorescence spectroscopy for detecting hazelnut oil adul-
tion sets, respectively. Quite similar results were obtained teration in virgin olive oils. Virgin olive, virgin hazelnut,
with AAA+NA scanned on egg yolks. The best results and refined hazelnut oil samples and a mixture of them at
were obtained with vitamin A fluorescence spectra, where 5%, 10%, 15%, 20%, 25%, and 30% adulteration were
correct classification rates of 97.7% and 85.7% in the analyzed after excitation at 350 nm. By performing linear
calibration and validation sets were obtained, respectively. discriminant analysis (LDA), 100% correct classification
The authors concluded that vitamin A fluorescence spectra was achieved. In a similar approach, Kyriakidis and
provide useful fingerprints allowing the identification of eggs Skarkalis (2000) used excitation wavelength of 360 nm to
during storage at low temperature and could be considered as differentiate between common vegetable oils, including olive
a powerful intrinsic probe for the evaluation of egg oil, olive residual oil, refined olive oil, corn oil, soybean oil,
freshness. Karoui and co-workers have continued this work sunflower oil, and cotton oil. All the oils studied showed a
by testing the ability of vitamin A fluorescence spectra to strong fluorescence band at 430–450 nm, except for virgin
monitor changes at the molecular level of 225 eggs stored at olive oil, which exhibited a low intensity at both 440 and
12.2 °C and 87% RH in an atmosphere containing 2% (n= 455 nm, a medium band around 681 nm and a strong one at
108) and 4.6% (n=99) of CO2 for 55 days (Karoui et al. 525 nm. The latter two bands have been ascribed to
2007e). Again, vitamin A fluorescence spectra allowed good chlorophyll and vitamin E compounds, respectively. The
discrimination of eggs according to both storage time and very low intensity of the peaks at 445 and 475 nm is due to
conditions, while more overlapping between egg samples the high content of phenolic antioxidants, which provide
was observed when the other intrinsic probes were investi- more stability against oxidation. All refined oils showed only
gated: eggs aged 22 days or less were separated from those one intense peak at 445 nm, which is due to fatty acid
aged 26 days or more. In addition, eggs were found to be oxidation products formed as a result of the large percentage
well separated for each storage time, except for those of polyunsaturated fatty acids present in these oils.
samples aged 20 and 22 days and those aged 26 and 29 days, Oil (and food systems in general) could be considered as
where some overlapping was observed. a complex system, which presents a set of different
Food Bioprocess Technol (2011) 4:364–386 379

properties and contains many fluorescent molecules. The describing the deterioration (e.g., anisidine value, iodine
use of only excitation or emission wavelengths could limit value, oligomers, and vitamin E).
the ability of this technique to determine the quality of oil
samples. To comply with this requirement, the variation in Cereals and Cereal Products
the excitation and emission wavelengths allows simulta-
neous determination of compounds present in oils. This The potential of fluorescence spectroscopy for monitoring
could be realized by using synchronous fluorescence cereals has increased over the past few years with the
spectroscopy. In this context, Sikorska et al. (2004, 2005) propagated application of chemometric tools and with
used synchronous fluorescence spectroscopy with excita- technical and optical developments of the spectrofluorim-
tion wavelengths from 250 to 450 nm and emission spectra eter. Zandomeneghi (1999) used FFFS (excitation 275 nm;
in the range 290 to 700 nm. The peak located at 320 nm emission 280–575 nm) to differentiate between different
after excitation at 290 nm has been attributed to tocopher- cereal flours (i.e., rice, creso, maize, pandas). The same
ols, while the band located at 670 nm in emission and research group also utilized visible excitation set at 445 nm
405 nm in excitation belongs to pigments of the chlorophyll (emission 460–600 nm) to differentiate between flours of
group. In order to compare the set of synchronous five different wheat varieties and a good discrimination was
fluorescence spectra of different oils, Sikorska et al. observed. In another study, excitation wavelengths set at
(2005) applied the k-nearest neighbors method, and good 275, 350, and 450 nm presenting fluorescence emission
discrimination between oil samples with a very low maxima at 335, 420, and 520 nm, respectively, were
classification error ranging between 1% and 2% and a low utilized to classify botanical tissue components of complex
standard deviation value was obtained. Guimet et al. (2004) wheat flour and rye flour; the bands were attributed to
explored the use of FFFS to discriminate between virgin AAA, ferulic acid, and riboflavin components, respective-
and pure olive oils; the ranges studied were at excitation ly. The last fluorescent component was confirmed by
wavelength λex =300–400 nm and emission wavelength Zandomeneghi et al. (2003), who attributed the band
(λem) 400–695 nm and λex =300–400 nm and λem 400– observed at 520 nm to riboflavin, while the band between
600 nm. The first range was found to contain chlorophylls, 430 and 530 nm was found to be proportional to the lutein
whereas the second range contained only the fluorescence content of the flour (Zandomeneghi et al. 2000).
spectra of the remaining compounds (oxidation products Ferulic acid and riboflavin spectra have been reported to
and vitamin E). Later, Guimet et al. (2005) applied have good accuracy for monitoring wheat flour refinement
PARAFAC to detect the adulteration of EVOO with olive- and milling efficiency following the use of fluorescence
pomace oil at low levels (5%). Discrimination between the imaging. Successful classification was obtained, suggesting
two types of oils was achieved by applying both LDA and that FFFS may be used to classify wheat cultivars (Symons
discriminant multi-way PLS regression; the latter method and Dexter 1991, 1992, 1993, 1994). These results have
gave 100% correct classification. The same technique recently been confirmed by Karoui et al. (2006a) where
(synchronous fluorescence) was used to analyze 73 sam- tryptophan fluorescence spectra of 59 samples (20 complete
ples, including 41 edible and 32 lampante virgin olive oils Kamut®, semi-complete Kamut®, and soft wheat flours, 28
collected in October and November 2002 (Poulli et al. pasta and 11 semolinas manufactured from complete Kamut®,
2005). PCA and hierarchical cluster analysis applied to the semi-complete Kamut®, and hard wheat flours) were scanned
emission spectra in the range 350–720 nm (at excitation after excitation at 290 nm. PCA performed on the flours’
wavelengths varying from 320 to 535 nm) showed good spectra clearly differentiated complete Kamut® and semi-
separation between the two types of oils. Recently, the same complete Kamut® samples from those produced from
research group assessed the potential of synchronous complete and semi-complete soft wheat flours, while good
fluorescence spectra to detect adulteration of virgin olive discrimination of pasta samples manufactured from complete
oil (VOO) with other oils (Poulli et al. 2007). By applying Kamut® and complete hard wheat flours from those made
PLS regression to the excitation spectra recorded 250– with semi-complete Kamut® and semi-complete hard wheat
720 nm with a wavelength interval of 20 nm, the authors flours was achieved. The best discrimination was obtained
stated that FFFS could be useful for the detection of olive- with tryptophan spectra recorded on semolinas, since the four
pomace, corn, sunflower, soybean, rapeseed, and walnut groups were well discriminated. Indeed, by applying FDA to
oils in VOO at levels of 2.6%, 3.8%, 4.3%, 4.2%, 3.6%, the spectral collection, 86.7% and 87.9% correct classification
and 13.8%, respectively. Frying oil deterioration has also rates were obtained for the calibration and validation samples,
been measured by using five selected excitation wave- respectively. In a similar approach, emission spectra (370–
lengths varying from 395 to 530 nm (Engelsen 1997). By 570 nm) were recorded after excitation at 350 nm on red and
applying PLS regression, the author showed good correla- white wheat kernels and a clear difference was observed
tion between fluorescence spectra and quality parameters between the two group samples (Ram et al. 2004); this
380 Food Bioprocess Technol (2011) 4:364–386

difference has been attributed to the morphological emission at 275 and 350 nm, respectively, was character-
variation in the pericarp and nuclear organization of the ized as an ultraviolet color precursor that participates in
two varieties of wheat. color development during storage. The other two (340,
420 nm and 390, 460 nm excitation/emission in the visible
Sugar wavelength area) are considered to be potential colorants,
which shows a link with their fluorescence behavior
In combination with multivariate statistical analyses, fluo- (Baunsgaard et al. 2000b). Recently, FFFS has been used
rescence spectroscopy has proved to be a promising to monitor adulteration of honey with cane sugar syrup
screening method for predicting quality parameters in beet (Ghosh et al. 2005). Using an excitation wavelength of
sugar samples (Munck et al. 1998). Indeed, it has been 340 nm, pure honey samples were characterized by two
shown that commercial sugars exhibit characteristic fluo- prominent features—a shoulder located at around 440 nm
rescence, which can be used to obtain information and a maximum located at 510 nm, which has been
regarding minor constituents in the sugar. Fluorescence ascribed to flavins—while cane sugar syrup samples
has successfully been applied to the beet sugar manufac- exhibited a maximum located around 430 nm. The peaks
turing process with the use of multivariate data analysis located at 440 and 430 nm in pure honey and sugar syrup
(Munck et al. 1998). The same approach with multiple samples have been attributed to NADH. Synchronous
excitation and emission wavelengths used by Carpenter and fluorescence was then applied to differentiate between pure
Wall (1972) has also been employed, and interesting results honey and sugar syrup samples, and good discrimination
were obtained. In a study of beet sugar samples, it was between the two groups was observed. The spectra for cane
possible to classify white sugar samples according to sugar syrup were characterized by a shoulder around
factory and to predict quality parameters such as amino 305 nm and a prominent band around 365 nm, while honey
nitrogen, color, and ash from the fluorescence data of these samples had a strong peak around 460 nm and a much
samples (Nørgaard 1995). The fluorescence data of thick weaker peak around 365 nm. The authors observed an
juice samples showed more ambiguous results owing to the increase in the intensity at 365 and 425 nm, as well as the
more complex sample composition. Another study of beet ratio of FI365/FI425, with the increase of cane sugar syrup
sugar samples utilized the three-dimensional structure of concentration; thus the ratio of FI365/FI425 has been
the fluorescence excitation–emission landscapes to resolve suggested as a potential method to monitor adulteration of
spectral excitation and emission profiles of fluorophores honey with cane sugar syrup. In another study, fluorescence
present in sugar with a multi-way chemometric model, spectra were scanned on 62 honey samples belonging to
PARAFAC (Bro 1999). Four fluorescent components were seven floral origins after excitation at 250 nm (emission
found to capture the variation in the fluorescence data of 280–480 nm), 290 nm (emission 305–500 nm), and 373 nm
268 sugar samples collected from a beet sugar factory in a (emission 380–600 nm) and emission set at 450 nm
single campaign, and two of them showed spectra with a (excitation 290–440 nm) by Ruoff et al. (2005) and Karoui
close similarity to the pure fluorescence spectra of the et al. (2007a). By applying FDA to the four data sets
amino acids tyrosine and tryptophan. The concentrations of (concatenation), correct classification rates of 100% and
the four components estimated from the sugar samples 90% were observed for the calibration and validation
could be correlated with several quality and process samples, respectively. In addition, the seven honey types
parameters, and they were characterized as potential were well discriminated, indicating that the molecular
indicator substances of the chemistry in the sugar process, environments, and thus the physicochemical properties, of
which has been confirmed by the use of HPLC analysis the investigated honeys were different. One of the main
combined with fluorescence detection on thick juice findings of these studies is that FFFS might be a suitable
samples and evaluation by PARAFAC (Baunsgaard et al. and alternative technique to classify honey samples accord-
2000a). Seven fluorophores were resolved from thick juice. ing to their botanical origins; this was confirmed recently
Apart from tyrosine and tryptophan, four of the fluoro- by Ruoff et al. (2006), who studied 371 honey samples
phores were identified as high molecular weight com- originating from Switzerland, Germany, Italy, Spain,
pounds, which were related to colorants absorbing at France, Slovenia, and Denmark. By using chemometric
420 nm. Three of the high molecular weight compounds tools, the error rates of the discriminant models ranged from
were found to be possible Maillard reaction polymers. The 0.1% to 7.5%.
last of the seven fluorophores indicated a compound with
polyphenolic characteristics. In a fluorescence study of 47 Fruit and Vegetables
raw cane sugars collected from many different locations
and campaign years, three individual fluorophores were Chlorophyll fluorescence has been used as an intrinsic
found; one of them, representing maximum excitation and probe to determine the physiological status of whole plants
Food Bioprocess Technol (2011) 4:364–386 381

and plant organs (Song et al. 1997). This component has recorded at different growth phases. By applying PCA to
been considered an efficient probe for monitoring apples the spectra scanned on each bacteria, three groups
during maturation, ripening, and senescence (Song et al. corresponding to three main phases of growth were
1997). Recently, fluorescence spectroscopy has been consid- identified (lag phase, exponential phase, and stationary
ered to have the potential for assessing the mealiness of phase). The authors then gathered the spectra recorded on
apples (Moshou et al. 2005), since relatively good correlation the three bacteria into one matrix, and this new matrix was
was obtained between mealiness and fluorescence spectra. analyzed by PCA. The obtained results showed good
Other authors have used chlorophyll fluorescence of apples discrimination of spectra according to bacteria and meta-
as a potential predictor of superficial scald development bolic profile. Recently, Ammor et al. (2004) utilized the
during storage (De Ell et al. 1996) and for the estimation of same technique for the identification of lactic acid bacteria
anthocyanins and total flavonoids in apples (Hagen et al. isolated from a small-scale facility producing traditional dry
2006). In another study, Lötze et al. (2006) used fluorescence sausages. Again, fluorescence spectroscopy demonstrated
imaging as a non-destructive method for the pre-harvest its ability to discriminate between Lactobacillus sakei
detection of bitter pit in apples; the same technique had subsp. carnosus and Lactobacillus sakei subsp. sakei. In
already been utilized to determine apple juice quality (Seiden another approach, Leriche et al. (2004) isolated 30
et al. 1996; Noh and Lu 2007). Two excitation wavelengths, Pseudomonas spp. strains from milk, water, cheese center,
set at 265 and 315 nm, were chosen as they yielded the and cheese surface belonging to two traditional workshops
richest spectra of two juice-apple varieties (Jonagold and manufacturing raw milk Saint Nectaire cheese. By applying
Elstar). The spectra showed two excitation–emission maxima FDA to the data sets, clear linkages between groups of
(315/440 and 265/350 nm) that have been not attributed to isolates were noted. In the first workshop, the milk was
any component in apple juice (Seiden et al. 1996). Applying implicated being as the sole source of cheese contamina-
PCA to the two juice-apple varieties, good discrimination tion, whereas in the second workshop the milk and cheese
was observed—which was not achieved with titratable center isolates were found to be similar, but different from
acidity or soluble solids data. The authors pointed out that surface cheese isolates. The authors attributed this contam-
an increase in the ripening process of apples involves an ination at the cheese surface to the water used during the
increase in the soluble fluorescent compounds. Good ripening process (washing of the cheese surface). From the
correlation between soluble solids and fluorescence spectra results obtained, it was stated that it is possible to
was observed independent of the apple varieties, indicating characterize, differentiate, and trace Pseudomonas spp.
the possibility of modeling the progression in maturity with strains using the fluorescence technique. These findings
information obtained from spectra, while fluorescence was were reinforced by the high correlation (using CCA)
found to correlate poorly to the amount of titratable acids in observed between the data sets obtained from the metabolic
juice. profiling and fluorescence spectroscopy. The obtained
results were fully supported by the same research group
Identification of Bacteria of Agro-alimentary Interest (Tourkya et al. 2009) since good discrimination, even for
strains for which ambiguity still remained after PCR and
The identification of microorganisms of agro-alimentary Analytical Profile Index 20 NE identification.
interest in food and food products by conventional Saito (2009) depicted that Napa cabbage (Brassica rapa
phenotypic procedures based on morphology and biochem- L.) laser-induced fluorescence (LIF) spectra of a normal
ical tests involves a large quantity of reagents and, in some core and a rotten core show greater intensities in the green
cases, is unable to discriminate microorganisms at the strain wavelength range of the LIF spectrum of the rotten core in
level. In this context, Leblanc and Dufour (2002) assessed comparison to that of the normal core. The integrated peak
the potential of different intrinsic probes (i.e., tryptophan, area between 450 and 600 nm for the rotten core was more
AAA+NA, and NADH) to discriminate between 25 strains than twice that for the normal core. Regarding pear (Pyrus
of bacteria in dilute suspensions. The best results were communis L.), differences were found in the LIF spectra
obtained by using AAA+NA spectra, where correct from the surface of a young pear and a ripe pear. The
classification rates of 100% and 81% were observed for intensity of the blue region (400–500 nm) of the LIF
the calibration and validation samples, respectively. The spectrum in the ripe pear surface was increased in
authors noted that fluorescence spectroscopy is able to comparison to the pear picked at harvest time in a still
discriminate and identify bacteria at genus, species, and unripe stage. In another approach, (Kim et al. 2003)
strain levels. This assumption was later demonstrated by the developed a LIF imaging food system (i.e., meat pork and
same research group (Leblanc and Dufour 2004). In their apple). Multispectral fluorescence emission images were
studies, the spectra of three bacteria strains (Lactococcus recorded after excitation set at 355 nm and the system
lactis, Staphylococcus carnosus, and Escherichia coli) were fluorescence emission images were captured in the blue,
382 Food Bioprocess Technol (2011) 4:364–386

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