Food Control 52 (2015) 103e111

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

Mid-infrared spectroscopy for discrimination and classification of

Aspergillus spp. contamination in peanuts
Hande Kaya-Celiker a, P. Kumar Mallikarjunan a, *, Archileo Kaaya b
a
Biological Systems Engineering, Virginia Tech, Blacksburg, VA, USA
b
Archileo Kaaya, Food Technology and Human Nutrition, Makerere University, Kampala, Uganda

a r t i c l e i n f o a b s t r a c t

Article history: In this study, Aspergillus spp., common colonists in peanut, were characterized, classified and quantified
Received 24 September 2014 using FTIR coupled with ATR accessory. FTIR-ATR spectral data of infected peanut samples were pre-
Received in revised form processed (mean-centering, smoothing the 1st derivative), and used for the PLS regression analysis for
11 December 2014
quantitative results. Very high R2 values (96.20e99.98%) together with low error of RMSEC values (0.014
Accepted 14 December 2014
Available online 23 December 2014
e0.153 Log CFU/g of peanut) were obtained. Even, the spectrum of peanut matrix was dominant at early
stages of invasion (
2.5 Log CFU/g peanut), resulting in section separation (Nigri from Flavi) and at higher
population (>4 Log CFU/g, species level separation (Aspergillus alliaceus, Aspergillus caelatus, Aspergillus
Keywords:
FTIR-ATR
flavus, Aspergillus parasiticus, and Aspergillus tamari) was observed. The accuracy of correct classification
Peanut increased proportionally with fungal invasion level and 100% correct classification was reached when the
Aspergillus species cell level was Log CFU/g ¼ 4.5e5. Samples with similar secondary metabolites (toxin producers) grouped
PLS regression close-by in PC score diagrams for all levels of fungal growth. Results highlight the possible imple-
Discriminant analysis mentation of FTIR-ATR model to detect infected peanuts even at early stages of invasion; besides, to
prove the potential separation capability in terms of species and their secondary metabolites.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cabanes, 1994), which is considered as a potent carcinogen in rats

(Mantle, Kulinskaya, & Nestler, 2005). A. flavus together with
The genus Aspergillus is ubiquitous in nature and distributed Aspergillus parasiticus are the most commonly seen opportunistic
worldwide. It is among the most studied of all fungal genera due to pathogen of crops and occur regularly in all-inclusive food supplies
their economic impact as being both industrially important bio- but especially seen in oil-rich seeds (Diener et al., 1987) like pea-
producer of certain enzymes and a causative agent in food nuts. Peanut is one of the most vulnerable crops to Aspergillus spp.
spoilage (Bennett, 2010). The genus contains about 250 species and contamination (Kaaya, Harris, & Eigel, 2006). Peanut pods are in
within the genus Aspergillus there are eight subgenera; and one or direct contact with soil, which is the source of primary inoculum for
more sections assigned to each subgenus (Raper & Fennell, 1965; Aspergillus spp., thus invasion of seed by these species before har-
Samson & Varga, 2010). Currently, there are 18 sections named, in vest is common (Horn, 2005). Other section Flavi species in peanut
total (Samson & Varga, 2010). Among them, section Flavi, which that have been reported are Aspergillus caelatus (Horn, 1997) and A
takes its name from infamous member, Aspergillus flavus, and the tamari (Horn et al., 1996), which are not aflatoxin producers; but
black aspergilli, Aspergillus niger have been frequently seen in Aspergillus tamari produces cyclopiazonic acid. Aspergillus alliaceus,
peanuts as dominant colonists. A. niger has always been important which have recently been placed in section Flavi (Peterson, 2008)
for biotechnology applications; for example, it's been used for has also been reported as being an ochratoxin A producer (Bayman,
synthesis of industrially valuable products like citric acid, or glu- Baker, Doster, Michailides, & Mahoney, 2002) and may colonize on
conic acid (Bennett, 2010). However, A. niger is also known for its peanut seeds depending on the environmental conditions of the
potential to produce ochratoxin (Abarca, Bragulat, Castella, & field (Horn, 2005).
Bearing the importance of mycotoxins in food and feed, there is
a need for rapid detection of toxic metabolites and their source
fungi; and many biochemical and molecular techniques are avail-
* Corresponding author. Tel.: þ1 540 231 7937; fax: þ1 540 231 3199.
E-mail addresses: [email protected] (H. Kaya-Celiker), [email protected] able today (Gilbert & Anklam, 2002). Fourier transform infrared
(P.K. Mallikarjunan), [email protected] (A. Kaaya). spectroscopy (FTIR) technology has gaining popularity in the area

http://dx.doi.org/10.1016/j.foodcont.2014.12.013
0956-7135/© 2014 Elsevier Ltd. All rights reserved.
104 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111

of identification and classification of microorganisms compared to pre-sterilized peanuts to obtain samples having varying cell con-
existing laborious and slow methods; and successful applications centrations of Aspergillus spp. of interest that will function as cali-
were built up even in subspecies level in years. Many authors have bration set. Paste was made for homogenous mold distribution by
reported the use of mid-infrared spectral data and chemometrics as grinding raw peanuts with a food processor (Butterfly Emerald
powerful discrimination tool for microorganisms and have been Mixer, Gandhimathi Appliances Ltd., Tamil Nadu, India) equipped
reviewed elsewhere (Mariey, Signolle, Amiel, & Travert, 2001). FTIR with metal cutting blade and stainless steel container. Initial fungal
in combination with reflectance accessorizes like attenuated total load was determined in terms of colony plate count and results
reflectance (ATR) (Kos, Lohninger, & Krska, 2003) or photoacoustic were expressed as averages Log CFU/g of peanuts of three mea-
spectroscopy (PAS) (Gordon, Jones, McClelland, Wicklow, & Greene, surements. Samples were kept in sealed containers and stored at
1999) have been proposed for Fusarium graminearum and A. flavus 4  C until analysis.
detection on corn, respectively. Recently, a research group in Ger-
many has shown successful usage of FTIR to identify airborne fungi
2.3. FTIR-ATR spectral measurements
and rapidly characterized “microfungi” stain (Fischer, Braun,
Thissen, & Dott, 2006). Fischer and colleagues have also demon-
The infrared spectra of contaminated peanut paste samples
strated different sample preparation protocols to get reproducible
were collected using FTIR spectrometer (Nicholet 6700, Thermo
procedure and repetitive spectral data of the living mycelia cells
Fisher Scientific Inc., Madison, WI, USA) coupled with DTGS KBr
(Fischer et al., 2006). In all these taxonomic classification studies,
detector. Sample compartment was equipped with Smart iTR dia-
including the bacterial cell identification (Naumann, 2000) quality
mond attenuated total reflectance (ATR) accessory (Thermo Fisher
of the developed multivariate regression or classification models
Scientific Inc., Madison, WI, USA) consisting of laminated diamond
depends on data compatibility, which was provided by standard-
as crystal material, mounted in stainless-steel plate. The diamond
izing the sample preparation procedures (Beekes, Lasch, &
crystal has a reflective index of 2.4 at 1000 cm1 and an angle of
Naumann, 2007). In this study, it was aimed to interpret spectral
incidence of 45 . Spectra were attained and processed with the
characteristics of Aspergillus spp. growing on peanut. It was pro-
Omnic Spectra (Thermo Scientific) software. Spectra were scanned
posed that different Aspergillus species have different growth pro-
over a range of 4000e625 cm1 at a resolution of 4 cm1 and a gain
file and different metabolism which can be detected by FTIR-ATR in
of 2.0. Average of 64 scans was used as final spectrum. Fourier
combination with multivariate analysis models. By doing so, sam-
transformation was performed with Mertz phase correction, NeB
ple preparation step was minimized (no need for isolation of fungi),
strong apodization function, with a zero filling factor of 2. The
peanut matrix was used as bulk medium and species classification
crystal was cleaned between successive measurements with 70%
was designed.
methanol and dried truly. The cleaned crystal was checked to avoid
residues from previous measurements by observing the back-
2. Methodology
ground spectrum. Three repetitive spectral readings were collected
per sample and to include the blocking effect, few of randomly
2.1. Aspergillus spp. spore solution preparation
selected samples were run again, by analyzing some of the samples
on the following day. All of the collected spectra were used for
Eight different strains, namely: A. alliaceus NRRL 4181,
analysis, not the averages. Samples prepared freshly (only stored
A. caelatus NRRL 25528, A. flavus NRRL 1957 (non aflatoxin pro-
during the course of the FTIR analysis) were spread onto diamond
ducing mutant), A. flavus NRRL 3357 (aflatoxin producer),
crystal until full contact was provided. Pressure knob was not used
A. parasiticus NRRL 21369 (non aflatoxin producing UV color
to avoid expressing oil content of paste upon pressure. Refrigerated
mutant), A. parasiticus NRRL 5862 (aflatoxin producer), A. tamari
samples were left at room temperature (23 ± 1  C) for 2 h to let
NRRL 20818 and A. niger NRRL 326 were kindly provided by Dr.
samples reach to room temperature before analysis.
Bruce Horn (USDA National peanut Research Lab., Georgia, USA).
Conidia of each strain from stock cultures were further inoculated
on potato dextrose agar and incubated at room temperature for 7 2.4. Multivariate data analysis
days to enable significant sporulation to take place. After incuba-
tion, 5 ml of sterile distilled water was aseptically added to each 2.4.1. PLS regression
plate. A sterile plastic inoculation loop was used to loosen the Partial least square (PLS) modeling is a powerful multivariate
colonies from the PDA plates. The suspension created was then analysis tool for spectral analysis of samples having complex
filtered through sterile cheese cloth into a sterile 50 ml capacity structure as is the case in peanut and mold itself. In this study,
Falcon tube. peanuts were infected with different strains of genus Aspergillus
and kept under high humid condition at room temperature to
2.2. Infecting peanut seeds with Aspergillus spp. accelerate the growth of fungi; and the spectral responses were
analyzed with PLS regression technique (TurboQuant IR calibration
Smooth, blanched, Virginia peanuts were obtained from local and prediction software, Thermo Fisher). Contaminated peanut
retailers and stored at 4  C in reclosable plastic bags until analysis. paste samples had cell concentration values varied between 2.5 and
Peanut pods were surface sterilized before inoculation treatment 6.0 Log CFU/g peanut. Readouts collected for each A. spp. were
using 1.0% NaOCl for 3 min and rinsed thoroughly with distilled 40e50 for calibration set and 20e24 for prediction set. Differences
water. After removing the excess water with pre-sterilized paper in the number of readouts were because some samples have more
towel, each pod was placed into petri-dish moist chamber (5 pods readings to include blocking effect. Spectral readings were pre-
per chamber, 2 chambers for each species), consisting of moist filter processed before loaded into PLS regression in order to reduce
paper to ensure high humidity (up to 80e100%). 10 ml spore sus- the variability associated with unrelated sources affecting the in-
pensions (105e106 CFU/ml) were dispersed onto surface sterilized tensity of all peaks. Data were mean centered to remove the de-
peanut seeds and were incubated in chamber at room temperature pendency on magnitude and filtered to smooth the peaks that
(23 ± 1  C) for 3 and 5 days to render contaminated peanuts at resemble random noise. First derivative of the data was filtered by
different levels of mold concentrations. Mold growth was visible Savitzky-Golay smoothing filter (data points ¼ 7 and polynomial
after 2 days. Seeds covered with masses of fungi were mixed with order ¼ 3).
 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111 105

The performance of developed PLS regression models for each with drierite. There are minor spectral differences within the species
Aspergillus spp. were tested by applying “leave one out” cross- of genus Aspergillus, but they all predominantly give intensive ab-
validation method in which each calibration standard was quanti- sorption bands between 3600e3100 cm1 and 1700e1500 cm1
fied as validation standard. The performance of developed PLS which are assigned to protein content (amide A, amide I and amide II
models were evaluated through correlation coefficient of deter- bands) of the fungi. If we briefly interpret the spectral profile, ab-
mination (R2), root mean-squared error of calibration (RMSEC), root sorption bands observed between 3050e2750 cm1 and
mean-squared error of cross-validation (RMSEV) and root mean- 1500e1300 cm1 are generally led by lipid content but there are also
squared error of prediction (RMSEP). some deformation modes of vibrations of protein structures. The re-
gion between 1200 and 900 cm1 is referred as ring vibrations of
2.4.2. Discriminant analysis oligo- and polysaccharides (Naumann, 2000) and region between 900
In order to determine the threshold cell concentration value and 600 cm1 is generally arising from aromatic ring vibrations (Fig.1).
which results in full discrimination of all Aspergillus spp. used in Since the early 1950s, there have been abundant of taxonomic
this study, samples were separated in four batches according to classification works published in the literature investigating
fungal growth level as samples having cell concentration of different spectral windows to get optimum dispersion of strains of
2.5 Log CFU/g peanut and lower, 3.0 to 4.0 Log CFU/g peanut, 4.5 to different bacterial or fungal cells (for review of early studies see
5 Log CFU/g peanut, and 5.5 Log CFU/g peanut and higher. Pre- (Mariey et al., 2001). Among many advantages of usage of infrared
processed (mean-centered, filtered by smoothing the 1st deriva- spectroscopy, analysis is limited to microorganisms that can grow
tive) spectral data of each batch was evaluated with respect to in culture media and stable and strictly controlled cultivation
quality of the classification using Discriminant analysis technique conditions (Naumann, 2000). Standardization of isolation of pure
(TurboQuant IR calibration and prediction software, Thermo Fisher) cultures from food matrix is one of the most time consuming step
by computing the distance from each class center in Mahalanobis for discrimination studies. When the fungal mycelium of Aspergillus
distance units. During calibration, the software computes a mean spp. was loaded to Discriminant analysis tool, at some extent,
spectrum and then generates a distribution model by estimating separation of different species were observed (Fig. 2). In this
the variance at each frequency in the analysis range. Results were analysis the spectral range of whole spectrum, 4000e650 cm1,
presented as principal component (PC) score plots. was used. Improving the model and standardizing the isolation
methodology, better discrimination of each species can be ach-
3. Results and discussion ieved. But, in this study, it was aimed to describe methodology that
reduces the sample preparation needs and do classification using
Peanut seeds were aseptically inoculated with spore solutions of the peanut matrix as it is; also, to depict the biochemical alterations
Aspergillus spp. and kept at humid environment until the masses of in both cell structure and nutritious medium (peanuts) as fungal
fungi of interest covered the seeds. Fig. 1 shows the average of 5 re- growth happens. Thus, spectral profiles of Aspergillus species (Fig. 1)
petitive reading of fungal mycelium of Aspergillus spp. scraped off the and the variation and correlation spectrum obtained from the PLS
peanut surfaces after drying infected peanuts in desiccator cabinet regression analysis (Fig. 3) were all used for spectral window

Mean spectrum of A.alliaceus NRRL 4181

Abs

0.2

0.4 Mean spectrum of A.caelatus NRRL 25528

Abs

0.2

0.4 Mean spectrum of A.flavus NRRL 1957

Abs

0.2

Mean spectrum of A.flavus NRRL 3357

Abs

0.2

0.4 Mean spectrum of A.niger NRRL 326

Abs

0.2

Mean spectrum of A.parasiticus NRRL 5862

Abs

0.2

Mean spectrum of A.parasiticus NRRL 21369

Abs

0.2

0.2 Mean spectrum of A.tamarii NRRL 20818

Abs

400 0 350 0 300 0 250 0 200 0 150 0 100 0

W a venum bers (c m -1)

Fig. 1. Mean spectra of each Aspergillus spp. studied. Mycelium of fungi of interest was scrapped off peanut surface, dried and loaded onto diamond cell of ATR unit for spectrum
collection.
106 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111

Table 1
Functional groups and vibration modes of FTIR spectral regions of fingerprint region
(Guillen & Cabo, 1997; Irudayaraj et al., 2001; Ismail et al., 1993).

Frequency (cm1) Functional group Mode of vibration

1743 eC]O (ester) Stretching

1710 eC]O (acid) Stretching
1643 C]O, CeN Stretching
1544 NeH, CeO Bending
CeC, CeN Stretching
1464 eCeH (CH2, CH3) Bending (scissoring)
1416 ]CeH (cis) Bending (rocking)
1378 eCeH (CH3) Bending (sym)
1238 eCeH (CH3) Bending (sym)
1160 eCeO, eCH2e Stretching, bending
1118 eCeO, eCH2e Stretching, bending
1095 eCeO Stretching

and the region representing the absorptions from the diamond

crystal (2340e1800 cm1) were excluded from all model calibra-
tions. Wave numbers between 3050 and 2750 cm1 were also
Fig. 2. Principal Component (PC) score plot for classes of different Aspergillus spp.
excluded from data analysis and the active portion of the spectrum
studied. ( ) A. niger NRRL 326; ( ) A. flavus NRRL 3357; ( ) A. flavus NRRL 1957; ( ) that lies between 1800 and 650 cm1 (fingerprint region) were
A. parasiticus NRRL 5862; ( ) A. parasiticus NRRL 21369; (◊) A. alliaceus NRRL 4181; (þ) used in the analysis. This region covers the variations that are un-
A. caelatus NRLL 25528; ( ) A. tamari NRRL 20818. ambiguous to fungal growth (Kos, Lohninger, & Krska, 2002).
Detailed band assignments of selected peaks in specified region
were summarized in Table 1. Representative spectral regions of
selection and interpretation of peaks. The variation and correlation
fungal growth on peanut were further illustrated in Fig. 4a, b and c,
spectra of each species of genus Aspergillus displayed regions that
and area alterations with respect to increased Log CFU/g peanut
are active and the association of these regions with respect to cell
values were indicated with arrows. Peanuts are complex structures,
concentration, respectively (Fig. 3).
with the main components being water (very little), proteins, fats
and carbohydrates. Proteins appear as amide I (1700e1600 cm1)
3.1. Spectral region selection and interpretation and amide II (1565e1520 cm1) groups (Stuart, 2004). The protein
related peaks were assigned to 1643 cm1 and 1544 cm1 in the
Given the complex structure of both Aspergillus spp. and peanut current study. The first one represents the carbonyl and CeN
matrices, the spectra can contain a superposition of hundreds of stretching of the amide I group and the second one relates to
infrared modes. The OeH stretching of water (3600e3200 cm1)

Fig. 3. PLS regression resulting statistical spectra: a: overall variance spectrum; b: multiple correlation spectrum; c: mean spectrum of infected peanut samples with A. flavus NRRL
1957 and d: mean spectrum of clean peanut samples.
 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111 107

Fig. 4. Spectral changes as A. flavus NRRL 1957 grows on peanut. Arrows show the direction of increasing A. flavus NRRL 1957 concentration (Log CFU/g peanut).

combination of the CeC and CeN stretching, and NeH and CeO band components were investigated. Same trend was noted for
bending vibrations of the amide II group (Irudayaraj, Sivakesava, amide A band (3290 cm1) but depth of concavity was smaller. It's
Kamath, & Yang, 2001). Another protein related band is usually been reported that proteolytic enzymes of fungi can hydrolyze the
observed around 3290 cm1 as a broad peak (NeH stretching of host proteins and can convert the building blocks into fungal pro-
amide group) but this region was not included in regression anal- tein during advanced stages of deterioration (Bothast, 1978). There
ysis. As stated before, fungal infection can be determined from the is also a host-fungus interrelationship, in which the level of protein,
increased absorption peaks of proteins, and decreased absorptions protease activity or amount of free amino acids differ significantly
at lipid and carbohydrate regions due to the metabolic activity of among the species of Aspergillus grown on peanut (Achar,
fungus (Naumann, 2000). In all Aspergillus spp. profiles, a sharp Sreenivasa, & Galdo, 2011); and only when heavily infected, free
decrease and a steady increase after cell concentration reaches to amino acid content shows a trend with respect to fungal growth
5 Log CFU/g of peanut was observed when amide I and amide II level (Chiou, 1997). Similarly, our results suggest fungal metabolism
108 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111

of peanut proteins were metabolized at first (concave down), and 1118 and 1095 cm1 decreased with increased mold growth. Those
then, fungal proteins started to be spectrally observable after a bands may be assigned to CeO group in esters (Guillen & Cabo,
certain amount of fungal growth (concave up). 1997) of triglycerides and can be used for estimating the level of
Along with protein peaks, the FTIR spectra of infected and clean lipid degradation or fungal growth. Differently, the peak at
peanut paste samples also showed a sharp linear increase and/or 1416 cm1 rose with half of the rate compared to previous bands in
decrease at fat associated peaks indicating lipid hydrolysis. Peanut regard to Aspergillus count.
is composed of mixed glycerides and contains several fatty acids, In our earlier studies, we tried to spike the peanut paste with
accounting for its high proportion of unsaturated structure (almost Aspergillus spore solution. However, no detectable, visible change in
80% oleic and linoleic acid content (Caballero, Trugo, & Finglas, spectrum was observed (data not shown), and peanut was domi-
2003); this structure is known to enhance the growth rate of nant in all aspects. Current results prove that metabolic activity of
Aspergillus spp. (Fanelli & Fabbri, 1980). Beside the growth rate, the fungus is inevitably the leading factor that affects the spectral
percentage of polyunsaturated fatty acids in peanuts affects the readings after certain level of infection is reached. Besides, different
amount of mycotoxin produced (e.g., results in induced aflatoxin species have different metabolism, different primary or secondary
production by lipoperoxides in A. parasiticus and A. flavus (Fabbri, metabolites, thus, different chemical profiles at different levels of
Fanelli, Panfili, Passi, & Fasella, 1983)). The enzymatic degradation growth. These chemical alterations were the basis of Discriminant
of lipids into free fatty acids and glycerol has been shown to be analysis and classification with respect to mold level.
prior to metabolism of starch granules or protein content (Smart,
Wicklow, & Caldwell, 1990), during Aspergillus invasion; alterna- 3.2. PLS model calibration and validation
tively, in some studies drop in sugar concentration was reported to
be before or simultaneous to triglyceride hydrolysis through PLS regression analysis is to predict dependent variables from a
metabolism of Aspergillus (Mellon, Cotty, & Dowd, 2000; Mellon, set of independent variables achieved by extracting a set of latent
Dowd, & Cotty, 2002). Still, hydrolysis of triglycerides by the ac- variables which have the best predictive power and to obtain a
tion of lipases has been an important alert to mold growth and relationship between the FTIR predicted and actual values. Separate
consequently increase in the free fatty acid content of grain has PLS regression models were developed for each species including
been suggested as an indicator of grain deterioration (Bothast, the cell concentration information in terms of Log CFU/g of peanut
1978). The peak height of the fatty acid ester linkage (eC]O) by deciding on the minimum number of factors accounting for as
centering at 1743 cm1 decreased due to lipid consumption and much of the manifest variation as possible while modeling the
simultaneously ester carbonyl (eC]O stretching of acid) of responses well. Pre-processed data (mean-centered, filtered by
breakdown product of free fatty acids (FFA) appeared and steadily smoothing the 1st derivative) was loaded into PLS (SIMPLE algo-
increased at 1710 cm1 (Ismail, Vandevoort, Emo, & Sedman, 1993). rithm) and resulted regression parameters were summarized in
Another fat associated band of CeO stretching at 1160 cme1 Table 2. All models fit the actual cell concentration values well with
generally decreased as mold count increased. These bands can be R2 values ranging between 96.20% (A. alliaceus NRRL 4181) and
used for determination of level of Aspergillus spp. contamination in 99.98% (A. flavus NRRL 1957); and the RMSEC values were between
peanut samples (Fig. 5), because all three gave a good correlation 0.153 and 0.014 Log CFU/g of peanut for the same species, respec-
with high coefficient of determination, R2 values of 98.21, 98.71, tively. The highest RMSEP, which is an indicator of the accuracy of
99.37% for A. flavus NRRL 1957 (as an example), respectively, when the model, was observed for A. tamari NRRL 20818 with a value of
linearly regressed to mold amount (similar trends were observed 0.235 Log CFU/g of peanut. When % difference was investigated, it
for other species, too). Apart from these peaks, other fat related was higher for prediction standards than the data matrices used for
peaks at 1464 and 1378 cme1, which represent bending vibrations calibration, which is probably a result of large factors number used.
of the methylene and symmetrical bending vibration of methyl This indicates an over-fit of calibration model developed for
groups of the fatty acids, respectively, showed a slight drop in A. tamari NRRL 20818. Similarly the highest RMSEV was found for
absorbance until mold counts between 20,000 and 100,000 cells; A. tamari NRRL 20818 (0.280 Log CFU/g of peanut), while A. niger
afterward increased to initial absorbance value as invasion NRRL 326 gave the lowest errors with values of 0.153 and
advanced. The height of peak at 1238 cm1, which related to the 0.120 Log CFU/g of peanut for RMSEV and RMSEP, respectively. The
CeO stretching of esters, had a similar trend, whereas the band at factor numbers were decided according to the predicted residual
error sum of squares (PRESS) plot. PRESS value decreases as the
factors are added. At some point error value reaches down to a
0.30 minimum, which gives the number of factors used in PLS. Adding
more factors will result in over-fitted calibration models. The
0.25
number of factors used for each species of Aspergillus was given in
Table 2 and actual versus predicted cell count values were illus-
Absorbance

0.20
trated in Fig. 6. In general, low levels of errors of calibration, vali-
0.15 dation and prediction data sets for all species studied prove the
high performance of FTIR when used as an analytical tool for esti-
0.10
mation of mold count. In other words, level of infection by Asper-
0.05 gillus spp. on peanuts can be determined with very little sample
preparation and without usage of chemicals in a very short time
0.00 (less than 1 min.).
0 100000 200000 300000 400000

Mold count (CFU/g) 3.3. Classification with respect to cell concentration

Fig. 5. Average of 3 absorbance readings with respect to cell count (CFU/g peanut) for Firstly, the whole data pool was separated into 4 batches (each
▫
A. flavus NRRL 1957 at specified wave numbers: (◊) at 1743 cm1; ( ) at 1710 cm1;
having 8 classes representing different Aspergillus spp.) and sepa-
(B) at 1160 cm1. The corresponding R2 values and the equations of fitting curves are:
▪
(dd) y ¼ 2*107x þ 0.2408 & R2 ¼ 0.9848; (d d) y ¼ 2*107x þ 0.02 & rate Discriminant Analyses were applied to investigate the
R2 ¼ 0.9879; (- - -) y ¼ 1*107x þ 0.1798 & R2 ¼ 0.9935. threshold cell count values to get full discrimination at species
 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111 109

Table 2
PLS regression statistics of each Aspergillus spp. studied.

Index Aspergillus spp. studied Factor Slope Intercept R2 (%) RMSEC (Log CFU/g) RMSECV (Log CFU/g) RMSEP (Log CFU/g)

1 A. niger NRRL 326 6 1.006 0.032 99.55 0.077 0.153 0.120

2 A. caelatus NRRL 25528 6 0.990 0.041 99.55 0.077 0.223 0.140
3 A. tamari NRRL 20818 9 0.983 0.045 99.90 0.035 0.280 0.235
4 A. flavus NRRL 1957 9 0.993 0.031 99.98 0.014 0.153 0.169
5 A. flavus NRRL 3357 5 0.977 0.098 98.96 0.117 0.246 0.189
6 A. parasiticus NRRL 5862 6 0.989 0.043 99.67 0.068 0.197 0.164
7 A. parasiticus NRRL 21369 6 0.994 0.034 99.65 0.068 0.1160 0.139
8 A. alliaceus NRRL 4181 3 0.972 0.094 96.20 0.153 0.244 0.195

6
FTIR Predicted Cell Count Values (Log CFU/g)

1
1 2 3 4 5 6
Actual Cell Count Values (LogCFU/g)
▵
Fig. 6. FTIR predicted versus actual cell count (Log CFU\g) values for all Aspergillus species studied: ( ) A. niger NRRL 326; ( ) A. flavus NRRL 3357; ( ) A. flavus NRRL 1957; ( )
A. parasiticus NRRL 5862; ( ) A. parasiticus NRRL 21369; ( ) A. alliaceus NRRL 4181; ( )A. caelatus NRLL 25528; ( ) A. tamari NRRL 20818.

level. Ten principal components were used to calibrate the method different metabolites produced by strains in the same taxon were
for all data sets; and 74.1, 76.5, 77.6, and 79.6 percent variability reported to be discriminated well (Fischer et al., 2006). Likewise,
were described for groups of Log CFU/g
2.5, Log CFU/g ¼ 3.0e4.0, the level and type of mycotoxin produced is related to the level of
Log CFU/g ¼ 4.5e5, and Log CFU/g  5.5, respectively. infection, and this was used as an indicator for discrimination and
The PC scores diagnostics for each group were illustrated in quantification of different Aspergillus spp. with respect to their
Fig. 7aed. PC scores of each class (Aspergillus spp.) had a tendency secondary metabolites (Kaya-Celiker, Mallikarjunan, Schmale, &
to depart from other classes as the cell concentration increases, as Christie, 2014). This can be a good explanation for A. niger
expected. When the cell counts were lower than 500, all species belonging to section Nigri getting separated first, even the cell
clustered close to each other, still a separation could be observed count was around 1000 CFU/g. Also, A. niger is known to produce
(Fig. 7a). Within the group of 2.5 Log CFU/g peanut and lower, 6 ochratoxin A. All other 7 species were from section Flavi, and strain
standards were misclassified; and misclassified sample number level separation required more cell count (around 10,000) to be
decreased to 3 for the group of 3.0e4.0 Log CFU/g peanut. For low fully distinguished.
level of Aspergillus invasion, it is obvious that the spectral changes
were predominantly from peanut matrix. But, after mold count of 4. Conclusion
1000 CFU/g was reached, first the A. niger NRRL 326 separated
which was followed by A. tamari NRRL 20818 and UV color mutant Both peanut matrix and the invaded fungi have complex
A. parasiticus NRRL 21369 (Fig. 7b). In groups of Log CFU/g ¼ 4.5e5, structures, and both are readily giving absorptions at similar fre-
and LogCFU/g  5.5 all sample data belongs to 8 species of genus quencies, or masking the spectral contributions of one another. At
Aspergillus were classified correctly (Fig. 7c and Fig. 7d). The most low level of fungal growth (Log CFU/g
2.5), peanut was found to
conspicuous observation was that for all cell concentration values be main component in spectral readouts. However, some compo-
the class centers of A. flavus 3357 and A. parasiticus NRRL 5862 sitional changes like a decrease and consequent increase at protein
remained close by. These strains are known to be an aflatoxin related peaks, or steady drop in lipid associated peaks indicated
producer, and apparently similar secondary metabolites produced that fungus of interest became dominant in the medium. PLS
increases the similarity within the spectral readouts. Previously, regression results prove the potential of FTIR-ATR as a tool for
110 H. Kaya-Celiker et al. / Food Control 52 (2015) 103e111

Fig. 7. PC score plot for groups: (a) Log CFU/g
2.5; (b) Log CFU/g ¼ 3.0e4.0; (c) Log CFU/g ¼ 4.5e5; (d) Log CFU/g  5.5. Aspergillus species are: ( ) A. niger NRRL 326; ( ) A. flavus
NRRL 3357; ( ) A. flavus NRRL 1957; ( ) A. parasiticus NRRL 5862; ( ) A. parasiticus NRRL 21369; (◊) A. alliaceus NRRL 4181; (þ) A. caelatus NRLL 25528; ( ) A. tamari NRRL 20818.

quantitative analysis of fungal growth on food matrix because very Achar, P. N., Sreenivasa, M. Y., & Galdo, G. (2011). Comparative studies on the
changes of total soluble proteins and protease activity in Georgian commercial
high R2 values together with low error percentages were found.
peanuts contaminated by Aspergillus flavus. Archives of Phytopathology and Plant
Besides, metabolic activity products of fungi added to spectral Protection, 45(2), 220e227. http://dx.doi.org/10.1080/03235408.2010.544465.
readings and it was observed that fungi in the same section were Bayman, P., Baker, J. L., Doster, M. A., Michailides, T. J., & Mahoney, N. E. (2002).
grouped together or split-up according to their secondary metab- Ochratoxin production by the Aspergillus ochraceus group and Aspergillus
alliaceus. Applied and Environmental Microbiology, 68(5), 2326e2329.
olites. This indicates that peanut kernels can be separated auto- Beekes, M., Lasch, P., & Naumann, D. (2007). Analytical applications of Fourier transform-
matically as healthy or invaded. Furthermore, according to the infrared (FT-IR) spectroscopy in microbiology and prion research. Veterinary
stages of deterioration by fungi, level of secondary metabolites may Microbiology, 123(4), 305e319. http://dx.doi.org/10.1016/j.vetmic.2007.04.010.
Bennett, J. W. (Ed.). (2010). An overview of the genus Aspergillus. Caister Academic.
be decided. Bothast, R. J. (Ed.). (1978). Fungal deterioration and related phenomena in cereals,
This project is funded by Peanut CRSP (USAID) aiming to legumes and oilseeds. Westport, CT, USA: Food and Nutrition Press, Inc.
improve livelihood of poor rural communities in East Africa by Caballero, B., Trugo, L. C., & Finglas, P. M. (2003). Encyclopedia of food sciences and
nutrition. Amsterdam: Academic Press.
emphasizing on health and nutrition aspects of aflatoxin and Chiou, R. Y. Y. (1997). Estimation of fungal infection of peanut kernels by deter-
Aspergillus infestation on peanuts. In many African countries, mination of free glutamic acid content. Applied and Environmental Microbiology,
especially in Uganda (where the project regionally focused on), 63(3), 1083e1087.
Diener, U. L., Cole, R. J., Sanders, T. H., Payne, G. A., Lee, L. S., & Klich, M. A. (1987).
there is a serious lack of trained personnel/scientist and up-to-date Epidemiology of aflatoxin formation by Aspergillus-flavus [Review] Annual Re-
technology to manage and analyze these contaminants on peanuts. view of Phytopathology, 25, 249e270. http://dx.doi.org/10.1146/
FTIR-ATR based technology is capable of rapid and non-destructive annurev.py.25.090187.001341.
Fabbri, A. A., Fanelli, C., Panfili, G., Passi, S., & Fasella, P. (1983). Lipoperoxidation and
measures of Aspergillus spp. very easily (analysis of one sample
aflatoxin biosynthesis by Aspergillus parasiticus and A. flavus. Journal of General
takes less than 1 min) and without the need for any harsh chemical Microbiology, 129(NOV), 3447e3452.
extractions. The method is not labor intensive as well. Potential Fanelli, C., & Fabbri, A. A. (1980). Growth requirements and lipid-metabolism of
implementation of proposed model can improve the quality of Aspergillus-flavus [Article] Transactions of the British Mycological Society,
75(DEC), 371e375.
peanuts supplied by means of sorting out the infected peanuts; and Fischer, G., Braun, S., Thissen, R., & Dott, W. (2006). FT-IR spectroscopy as a tool for
subsequently, conquer the health and trade constraint resulting rapid identification and intra-species characterization of airborne filamentous
from fungal contamination. fungi [Article] Journal of Microbiological Methods, 64(1), 63e77. http://
dx.doi.org/10.1016/j.mimet.2005.04.005.
Gilbert, J., & Anklam, E. (2002). Validation of analytical methods for determining
Acknowledgments mycotoxins in foodstuffs [Article] Trac-Trends in Analytical Chemistry, 21(6e7),
468e486. http://dx.doi.org/10.1016/s0165-9936(02)00604-0.
Gordon, S. H., Jones, R. W., McClelland, J. F., Wicklow, D. T., & Greene, R. V. (1999).
Authors wish to express their gratitude to Peanut CRSP program of Transient infrared spectroscopy for detection of toxigenic fungus in corn: po-
USAID for providing the financial support to the project. Authors also tential for on-line evaluation. Journal of Agricultural and Food Chemistry, 47(12),
5267e5272. http://dx.doi.org/10.1021/jf990011f.
would like to extend their gratitude to Drs. Maria Elisa Christie, Justin Guillen, M. D., & Cabo, N. (1997). Characterization of edible oils and lard by Fourier
Barone and Charity Mutegi for their participation in the project. transform infrared spectroscopy. Relationships between composition and frequency
of concrete bands in the fingerprint region. Journal of the American Oil Chemists
Society, 74(10), 1281e1286. http://dx.doi.org/10.1007/s11746-997-0058-4.
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