Original research article

Journal of Near Infrared Spectroscopy

2021, Vol. 29(2) 63–72
Two-dimensional mid and near infrared ! The Author(s) 2020
Article reuse guidelines:
correlation spectroscopy for bacterial sagepub.com/journals-permissions
DOI: 10.1177/0967033520974518
identification journals.sagepub.com/home/jns

Pavel Krepelka1 , Araceli Bol
ıvar2 and Fernando P
erez-Rodr
ıguez2

Abstract
In recent years, near infrared (NIR) spectroscopy has gained interest as a tool for bacteria strain identification. Although
some promising results suggest good applicability of the technique, a better interpretation of the NIR bacterial spectra is
still needed. In order to analyze the NIR spectrum of biological samples, a correlation analysis between the NIR and the
mid-infrared (mid-IR) spectra was performed. In total, 28 spectra of 8 bacterial strains were acquired and correlated in the
NIR and the mid-IR spectral ranges. Some molecular bands (Amide I, P ¼ O stretching, C-H stretching/deformation of
polysaccharides) were well correlated, and the effect of concentration changes in these molecules were investigated.
Moreover, a model for the NIR spectra classification was created with an overall 85% correct classification rate.
Subsequently, only NIR wavelengths with high correlation to important mid-IR peaks were selected. This led to an increase
in the correct classification rate to 94%. By correlation between well-established mid-IR peaks and NIR spectra, some
relationships in the NIR spectra of biological samples were revealed, which was a step towards better understanding and
interpretation of the NIR spectra of biological samples.

Keywords
Bacterial identification, classification, near infrared, mid infrared, correlation, 2D correlation, microbiology
Received 8 May 2019; accepted 3 July 2020

Introduction
Infrared spectroscopy is a widely used analytical tech-
nique exploiting light absorption by specific molecu-
lar bonds. The light absorption by the examined
sample is wavelength-selective, and therefore the pres-
ence of many chemical substances can be predicted
according to the acquired infrared absorption spec-
trum.1 Although the absorption peaks observed in the
mid-infrared (mid-IR) range (2.5 mm–25 mm) arise
from fundamental molecular vibration modes and
are relatively easy to separate and interpret, peaks
attributable to the NIR (near infrared) spectrum
(1000–2500 nm) consist of overtones and combination
modes and are 10–100 times weaker and overlapped.2
This makes the NIR spectrum difficult to interpret.
From this reason, precise and clear information of the
composition of the analyzed sample is very problem-
atic to obtain.
Mathematical treatment, including derivatives,
smoothing, and scatter correction, is routinely utilized
to treat the spectra and enhance spectral details. As
the spectral variables are highly collinear, calibration
models to reveal the dependencies between the NIR
spectra and the predicting variables must be
computed by using multivariate statistics. The most
widely used models are linear ones (PCA, PLS,
SIMCA),3–5 and for complex applications, nonlinear
models (SVM, ANN)6 often exhibit better results.
Creating large calibration sets usually requires a sub-
stantial amount of time and resources; calibration
models are therefore mostly built on a limited dataset
and must be validated very carefully. Insufficient val-
idation presented in numerous publications delivers
misleading chemical analysis of the investigated sam-
ples and mistaken conclusions of the suitability of
NIR spectroscopy. More precise assignment of NIR
spectral bands to specific chemical constituents could
1
Department of Theoretical and Experimental Electrical Engineering,
Brno University of Technology, Brno, Czech Republic
2
Department of Food Science and Technology, Faculty of Veterinary,
Agrifood Campus of International Excellence (CeiA3), University of
Cordoba, C
ordoba, Spain
Corresponding author:
Pavel Krepelka, Department of Theoretical and Experimental Electrical
Engineering, Brno University of Technology, Technicka 12, 616 00, Brno,
Czech Republic.
Email: pavel.krep@gmail.com
64
enable to improve the accuracy of calibration models
for NIR spectroscopy.
While mid-IR spectroscopy became well estab-
lished in microbiology,7 its NIR counterpart found
use mainly in the food industry and within food
safety applications, some of the reasons being lower
sensitivity to water, the high scan speed (real-time
analysis), simpler and cheaper instruments, non-
destructive methodology and minimal sample prepa-
ration requirements. Alexandrakis et al.8 investigated
bacterial contamination in chicken breasts. With NIR
spectroscopy and multivariate calibration, breasts
exhibiting bacterial concentrations higher than 6 log
CFU g1 (colony-forming units per gram) were suc-
cessfully identified. The study8 established that satis-
factory identification via NIR spectroscopy was a
consequence of the proteolytic process, with a
higher absorption in the amide and amine bands;
such a property of the technique would indicate aug-
mented concentration of amino acids and low-
molecular peptides during microbial spoilage of
meat. Spectral bands associated with molecular
bonds in proteins, peptides, amides, and amino
acids (1864–2100 nm; 1396–1550 nm; 1386–1570 nm;
1856–2089 nm) were identified by Alexandrakis et al.8
Probably the most significant progress in bacteria
identification via NIR spectra was achieved by
Rodriguez-Saona et al.,9 who eliminated the effect
of water by filtering the bacterial suspension with
filter membranes made of an inorganic material
(Anodisc aluminum oxide). Pure spectra of bacterial
biomass were acquired, and the authors proposed a
partial interpretation of spectral peaks based on addi-
tional measurement of isolated cellular components
(deoxyribonucleic acid standard, glycogen standard,
lecithin standard, and N-acetylmuramic acid stan-
dard). When a sufficient number of bacteria (i.e., bio-
mass) had been deposited on the filter (10 mg of wet
pellets), separable clusters were obtained in the PCA
scores plot, corresponding to six bacterial species.
Thus, the potential of NIR spectroscopy for bacteria
identification was confirmed. In a later study,
Rodriguez-Saona et al.10 employed the same proce-
dure to discriminate bacteria extracted from juice. By
using the region between 1920–2500 nm, the authors
observed good clustering in the score plot according
to the bacterial species. This was achieved by depos-
iting only 1 mg of wet pellets onto the membrane,
together with ethanol application.
Dubois et al.11 demonstrated the potential of NIR
spectroscopy for bacteria analysis by selecting spec-
tral wavelengths (1200 nm; 1680 nm; 1734 nm;
1765 nm; 2150–2190 nm; 2310 nm; and 2345 nm)
which originate mainly from fatty acids and contrib-
ute substantially to bacteria identification. In other
research reports, NIR spectroscopy, combined with
multivariate statistics, was applied to quantify and
classify microbial contamination in food products,
such as flounder12 and chicken fillets,13 chicken
Journal of Near Infrared Spectroscopy 29(2)
breasts,8 salmon,14 pork meat,15 juice,10 and
cheese.16 Most of these studies identify spectral
bands significant for classifying or quantifying bacte-
rial strains in food; however, interpretation of the
spectra is generally tentative and imprecise as regards
the molecules and compounds responsible for micro-
bial identification and discrimination. Additionally,
results vary between studies, due to the above-
mentioned limitations of NIR spectroscopy.
By contrast, the mid-IR spectrum of bacterial bio-
mass is well interpreted. The most intensive investiga-
tion targeting the discussed problem appears to have
been performed by Helm17 and Naumann,7,18 who
proposed assignment of spectral bands found in the
mid-IR spectra of bacteria. In total, 13 bands were
assigned, and this interpretation was used for the cor-
relation experiments.
Numerous papers on bacteria strain identification
in the mid-IR range have been published to date, and
foodborne pathogenic bacteria in particular has
attracted broad attention. In this context, for exam-
ple, Janbu et al.19 achieved the success rate of 100%
in the classification of Listeria (5 species, 30 strains).
Al-Holy et al.20 recognized 4 foodborne pathogens
(Bacillus cereus, Salmonella enterica, Escherichia
coli, and Listeria spp.) measured on aluminum oxide
filters at the concentration of 105–106 CFU mL1
with 94% correct classification rate. An exhaustive
list of publications discussing foodborne bacteria
identification is available in a relevant article by
Davis and Mauer.21 The popularity of mid-IR in
the identification of microorganisms shows itself
also in the FT-IR microplate reader, a device
designed to acquire mid-IR spectra from dried sus-
pensions and to carry out the subsequent classifica-
tion (Bruker HST-XT).
Improved interpretation of the NIR spectra of bac-
teria could contribute toward increasing the applica-
bility of NIR spectroscopy in microbiology. NIR/
mid-IR correlation spectroscopy, which compares
changes in the NIR and the mid-IR spectra of various
samples, is proposed as a powerful tool to determine
NIR bands and associated chemical bonds and mol-
ecules accounting for bacterial identification and
quantification, with mid-IR employed as the
benchmark.
Materials and methods
Bacterial strains and preculture conditions
The microorganisms used in this study were obtained
in the lyophilized form from the Spanish Type
Culture Collection (Valencia, Spain) and reconsti-
tuted following the provider’s recommendations.
The cultures were maintained at –18 C in cryogenic
vials (MicrobankTM, Prolab Diagnostic, Neston,
UK). Eight bacterial strains were studied, including
Lactobacillus sakei (CECT 4808), Lactobacillus
Krepelka et al.
plantarum (ATCC 8014), Lactococcus lactis ssp.
Lactis (ATCC 19435T), Listeria monocytogenes
(NCTC 11994), Listeria ivanovii (ATCC 19119), and
Staphylococcus aureus (ATCC 23235) as gram-
positive bacteria, with Salmonella enteritidis (CECT
556) and Escherichia coli serotype O25:H42 (CECT
686) representing the gram-negative category.
The cultures were activated in different broth
media and at incubation temperatures according to
the optimum growth conditions of each microorgan-
ism, that is, de Man, Rogosa and Sharpe broth
(MRS, Oxoid, Basingstoke, UK) for lactic acid bac-
teria at 33  C in 10% CO2 (Memmert, Schwabach,
Germany); Brain Heart Infusion broth (BHI,
Oxoid) for L. monocytogenes, L. ivanovii, and S.
aureus; and Tryptone Soya Broth (TSB, Oxoid) for
Salmonella and E. coli at 37  C in an aerobic environ-
ment. For every experiment and microorganism, a
fresh purity plate was prepared from frozen stock
on a Plate Count Agar plate (PCA, Oxoid) incubated
for 24 h at the above-mentioned conditions. Two days
before the experiment, a well-isolated single colony
from the PC plates was transferred into a tube con-
taining 9 mL of the corresponding broth medium and
incubated under static conditions for 24 h at the same
temperatures. Then, 1 mL of each initial subculture
was refreshed in the corresponding broth and incu-
bated under identical conditions for 16 h.
Experimental design and filter membrane
preparation
To analyze the bacteria biomass spectra, a modified
version of the protocol by Rodriguez-Saona et al. [21]
was followed. For each bacterial strain, 1 mL of the
16 h-preculture was pipetted into a flask containing
150 mL of the corresponding growth medium and
incubated at the same conditions for the total
period of 48 h. The initial concentration in the inoc-
ulated flasks corresponded to ca. 107 CFU mL1.
This cell level was also used in a previous assay to
monitor microbial growth by optical density (OD)
measures in an automated plate reader (Bioscreen
C, Labsystems, Finland) under growth media and
temperature conditions identical with those described
in previous section. Based on the obtained OD curves,
four different growth phases were independently col-
lected for each microorganism from the inoculated
flasks at appropriate time intervals. The stages were
as follows: early exponential phase (batch 1), between
exponential and stationary phase (batch 2), late sta-
tionary phase (batch 3), and decline phase (batch 4).
To execute the actual collection procedure, culture
samples of 15 mL of broth were taken from the incu-
bated flasks and transferred into a 20-mL sterile cen-
trifuge tube. The bacterial cells were twice-washed in
0.85% Saline Solution (SS, Panreac, Barcelona,
Spain) by centrifugation at 4000 rpm (Jouan C4i;
65
Thermo Electron Corporation, Illkirch, France) per-
formed at room temperature for 15 min.
To eliminate the impact of the different bacterial
counts on the spectral measurements, the cell concen-
tration present at the lag phase was used in all the
batches. For this purpose, appropriate dilutions of
the washed suspensions were made for batches 2, 3,
and 4 by using sterile SS. The bacterial cells were
retained on glass microfiber filters (GF-5, 25 mm
diam., Whatman GF/A//WhatmanTM, Fisher
Scientific, UK) through vacuum filtering, utilizing a
Millipore Sterifil Aseptic System and Swinnex filter
holders (Millipore Corp., USA). The filter mem-
branes were dried for 1 h at 45  C.
Spectral acquisition
The NIR spectra were obtained in the diffuse reflec-
tance mode using the integrating sphere of an FT-
NIR spectrometer (Bruker MPA, Bruker Optics,
Ettlingen, Germany) having a PbS detector. The bac-
teria were analyzed directly by placing the membranes
on the top of the sphere. The spectra were collected
with a spectral resolution of 16 cm1 in the range of
12,500–3600 cm1 (800–2777 nm). Each spectrum was
recorded as the average of 32 scans over 772 data
points. The acquired NIR spectra are displayed in
Figure 2.
The mid-IR spectra were obtained via diffuse
reflectance using an FT-IR spectrometer (Bruker
Tensor 27, Bruker Optics, Ettlingen, Germany) spec-
trophotometer with a CsI beamsplitter and a DTGS
detector. The spectra were collected with a spectral
resolution of 4 cm1 over the range 4000–250 cm1.
Each spectrum was recorded as the average of 50
scans over 1944 data points. The acquired mid-IR
spectra are displayed in Figure 1.
For acquisition of both the NIR and the mid-IR
spectra OPUS v.6.5 software (Bruker Optics) was
employed. In total, 28 spectra of 8 bacterial strains
were collected (3 spectra of L. plantarum and L. sakei,
2 spectra of L. monocytogenes, and 4 spectra of
remaining strains). Since the goal of experiment was
to correlate NIR and mid-IR spectra, the number of
measurements should be sufficient for mutual NIR/
mid-IR variability. This contrasts with standard
regression or classification problems, where the
number of samples must be chosen to ensure high
variance of specific NIR/mid-IR wavelengths (vari-
ance that can be lowered by larger sample size).
Spectra processing
Chemometric data analysis was performed using
MATLAB R2016a (MathWorks, Natick, MA,
USA) with routines created in-house. In order to
enhance hidden and overlapped peaks, all the spectra
were treated with a Savitzky-Golay filter (2nd deriv-
ative; 3rd order polynomial; window: 31 points for
66 Journal of Near Infrared Spectroscopy 29(2)

Figure 1. Raw mid-IR spectra.

Figure 2. Raw NIR spectra.

mid-IR and 17 for NIR). The processed spectra are • 28 NIR and 28 mid-IR spectra of different bacte-
shown in Figure 3 and 4. rial strains were acquired.
• The dynamic spectra were computed by subtract-
Correlation map ing the average spectrum.

For the data correlation, two-dimension (2 D) cor- Ag

NIR ðvn ; iÞ ¼ ANIR ðvn ; iÞ  ANIR ðvn Þ (1)
relation spectroscopy was used.22 The resulting
2 D map shows the correlation between corre-
sponding NIR and mid-IR wavelengths. High pos- Ag
MIR ðvm ; iÞ ¼ AMIR ðvm ; iÞ  AMIR ðvm Þ (2)
itive values express a high direct proportion,
whereas high negative ones denote a high inverse Aðvn ; iÞ vn -th wavenumber of the i-th spectrum
proportion. Values close to zero indicate low cor- A ðvn Þ average spectrum
relation. The correlation spectrum was created as eðvn ; iÞ dynamic spectrum
A
follows:
Krepelka et al. 67

Figure 3. Mid-IR spectra after applying 2nd derivative Savitky-Golay filter + SNV.

Figure 4. NIR spectra after applying 2nd derivative Savitky-Golay filter + SNV.

• The synchronous correlation spectrum Uðvn ; vm Þ Bacteria strain classification model

represents the degree of correlation between
In the following portion of the paper, identification of
the NIR wavenumber vn and the mid-IR wavenum-
bacterial strains according to the NIR spectra is
ber vm .
described. For this purpose, the total of 90 spectra
1 Xg
p
Uðvn ; vm Þ ¼ ANIR ðvn ; iÞ:Ag
MIR ðvm ; iÞ (3) of three bacterial species (L. ivanovii ATCC 19119,
p  1 i¼1
S. enteritidis CECT 556, and E. coli O25:H42 CECT
686) were collected (30 for each strain). The micro-
Additional information is presented in a relevant organisms and filter membranes were prepared as
study by Noda.22 All equations (1-3) necessary to outlined in section “Experimental design and filter
construct the correlation map were implemented in- membrane preparation”.
house using MATLAB R2016a and the final map was The NIR spectra were acquired using a Nicolet
displayed using the function “surf”. Antaris II Fourier-transform spectrometer (Thermo
68
Fisher Scientific, Waltham, MA, USA) in the diffuse
reflectance mode with an integrating sphere. The data
were collected with 1 cm1 spectral resolution in the
range 9090–4000 cm1 (1100–2500 nm) with an average
of 60 scans. The spectral processing-related and addi-
tional information can be found in a previous study.23
The NIR spectra were classified using partial least
squares discriminant analysis (PLS-DA).3 The model
was validated via two-fold cross-validation.23
Consequently, the NIR wavelengths exhibiting high
correlation (r > 0.5) with mid-IR peaks (see Nauman7
for a detailed list). Because classification of bacterial
strains based on mid-IR spectrum is more accurate
than models based on NIR spectrum, the selection
of NIR spectral features strongly correlated with
mid-IR peaks should increase the classification per-
formance. However, broad information distribution
in NIR region do not allow strong correlation, that
is why the threshold 0.5 was chosen. Table 1 provides
the list of some of the peaks important for correla-
tion, as found by Nauman.7
Results and discussion
The correlation map shown in Figure 5. displays the
degree of correlation between the NIR (X axis) and
the mid-IR (Y axis) spectra. The brighter parts exhibit
stronger relationship between the NIR and the mid-
IR wavenumbers.
Table 1. Main mid-IR peaks of bacterial sample.7
Wavenumber Wavelength
P ¼ O stretching 1263 cm1
7918 nm
Amide II band 1548 cm1 6460 nm
Amide III band 1240–1310 cm1 7634–8065 nm
Amide I band 1695 cm1 5900 nm
CH2 in fatty acid 2924 cm1 3420 nm
Figure 5. Uðvn ; vm Þ NIR and mid-IR correlation map. Brighter
color represents higher degree of correlation.
Journal of Near Infrared Spectroscopy 29(2)
In the mid-IR spectra (the Y axis in Figure 5), a
highly correlated region is observed in the range
between 1000–1800 cm1. According to sources,7,21
the peaks in this region correspond to the vibrations
of proteins in the Amide group, O-H bending, CH2
bending, and C-H stretching in polysaccharides.
Another highly correlation region can be seen at
around 3000 cm1, where peaks interpreted as CH2
and CH3 stretching in lipids are located.
The region of the NIR spectra with the highest
correlation is found in the range from 4000–
5500 cm1 (1818–2500 nm) (the X axis in Figure 5).
Absorption at these wavelengths is attributed to
strong C-H, N-H, O-H combination bands. Another
significant correlation region lies between 7000–
7500 cm1 (1333–1428 nm), corresponding mainly to
the 1st overtone of the C-H combinations.
In the mid-IR spectrum, the peaks resulting from the
vibrations in Amide molecules are easily distinguishable
at the wavenumbers of 1695 cm1, 1685 cm1,
1675 cm1, 1655 cm1, and 1637 cm1 for Amide I
and 1548 cm1 for Amide II; the peaks in the range
of 1310–1240 cm1 are associated with Amide III.
Figure 6 shows the correlation with one of the
Amide I peaks (1695 cm1) within the whole NIR
spectra. Higher (positive/negative) values represent a
higher (direct/inverse) proportion with the Amide I
peak. Hecht et al.,24 Martis et al.,25 and Rodriguez-
Saona et al.26 recognized these peaks associated with
Amide I, as shown in Table 2.
As shown in Figure 6(a), these peaks indeed exhib-
ited high correlation values. The peaks were correlat-
ed with the mid-IR Amide I peak, containing
information about this functional group. Moreover,
very high correlation was observed in the peak at
wavenumber 4332 cm1 (2308 nm), belonging to the
P ¼ O bond. The Amide II (1548 cm1) and Amide III
peaks (1310–1240 cm1) exhibited the same correla-
tion as Amide I, showing that these bonds appeared
to be inseparable according to the NIR spectra.
A good value (r ¼ 0.2–0.4) for the P ¼ O stretching
peak was observed at 1263 cm1 (Figure 6(b)). Two
distinctive peaks on locations 4332 cm1 (2308 nm)
and 4258 cm1 (2348 nm) dominated the correlation
plot. Rodriguez-Saona et al.26 identified these peaks
via measurement of phospholipids (e.g., lecithin). The
P ¼ O stretching vibration mode can be very reliably
detected by NIR spectroscopy. The peak associated
with the Amide group also occurred in correlation
with the P ¼ O stretching peak, which could be
caused by the chemical structure of membrane com-
plex lipids such as sphingolipids.
Figure 6(c) shows correlation of the NIR spectrum
with a peak that Naumann7 identified as being
responsible for the C-H asymmetric stretching of
methylene (CH2) in fatty acids. As predicted, the
correlation contains multiple peaks referred to in
the literature, particularly the region between
4000–5000 cm1 (2000–2500 nm). The list of
Krepelka et al. 69

Figure 6. Correlation coefficient of NIR wavenumbers with (a) Amide I peak (1695 cm1), (b) with P ¼ O stretching peak (1263 cm1) and
(c) CH2 in fatty acid (2924 cm1).

interpreted peaks related to bacterial cell classification numerous publications as a result of the C-H stretch-
is shown in Table 2. ing/deformation of polysaccharides.2,26
Very strong correlation (r ¼ 0.75) was found at Not all of the mid-IR peaks identified by
4294 cm1 (2329 nm). This peak is considered in Naumann et al.18 are seen to be correlated with the
70 Journal of Near Infrared Spectroscopy 29(2)

Table 2. NIR absorbances with high correlation to Amide I and to CH2 in fatty acid.

Functional group Band assignment Wavelength (nm) Wavenumber (cm1)

Amide I Amide A/I and Amide B/II26 2052–2060 4854–4874

Water bonded to protein24 1945 5141
Protein content25 1520 6579
CH2 in fatty acid C-H stretching/deformation26 2366 4226
Amino acid combination bands10 2134–2233 4478–4686
O-H stretching/deformation 2080 4808
C ¼ O, carbonyl overtone in fatty acid10 2033 4920

Table 3. The data set preparation and performance of the PLS-DA model.

Whole spectra Shortened spectra

PLS-factors for prediction 3 3

Pre-processing Second order (SG + SNV) Second order (SG + SNV)
N calibration 45 45
N validation 45 45
CCR of calibration (%) 88 96
CCR of validation (%) 85 94
Validation method 2-fold CV 2-fold CV
N: number of samples; SG: Savitzky-Golay filter; SNV: standard normal variate; CRR: correct classification rate; 2-fold CV: 2-fold
cross validation. Original sample is randomly partitioned into 2 equal sized subsamples. First subsample is retained as the
validation data and second subsamples are used as training data.

Figure 7. Partial least squares discriminant analysis (PLS-DA) score plots of whole and shortened NIR bacterial spectra.

NIR spectra; the C ¼ O and C-O molecule bonds in
particular have a small impact on the NIR spectra
because of the low absorption in this region.
As regards direct utilization of NIR spectroscopy
in classifying or analyzing bacterial cells, it is impor-
tant to note that the results in this study are derived
from experiments performed at a relatively high cell
concentration (~107 CFU mL1) and under static
growth conditions. Environmental factors, preculture
conditions (e.g., nutrients availability or incubation
temperature, culture medium), and the growth stage
all affect the acquired spectra; these factors need to be
considered in setting up the classification model.
For analysis of NIR vs. mid-IR correlation, the var-
iability that is not related to strains is acceptable since
the variability is caused by diversion in chemical com-
position that is similar to diversion in spectra of var-
ious strains.
Nevertheless, the fact that the most important mid-
IR spectral bands linked to the major bacterial cell
constituents are correlated on the NIR spectra sug-
gests the possibility of reliable interpretation of the
NIR spectrum even at lower concentration levels.
To illustrate how the outcomes of this study are
applicable in NIR model generation, the entire NIR
spectra of 3 bacterial species were used to construct
Krepelka et al.
PLS-DA classification model. The correct
classification rate (CCR) of this model is available
in Table 3 (61%).
To improve the NIR model performance, a new
model was constructed in this study, though
only based on wavelengths with a high correlation
rate (|r|>0.5). These spectra with selected wavelengths
were used for classification, and the correct classifica-
tion rate increased to 96% (Table 3). For comparison,
a PLS-DA score plot is shown in Figure 7. Better
separability is evident in the score plot of the model
built on selected wavelengths. As can be observed, the
NIR vs. mid-IR correlation enabled the selection of
the best NIR wavelengths for bacteria identification.
Conclusion
The use of NIR spectroscopy in bacterial cell analysis
was assessed by a two-dimensional NIR vs. mid-IR
correlation analysis. The results confirmed that the
most significant NIR spectral variations are associat-
ed with molecule bonds found in major classes cell
macromolecules, i.e., proteins, phospholipids, and
polysaccharides. For instance, amide groups are
located in cellular proteins, which represents about
40–60% cell dry weight.7 Phospholipids and polysac-
charides are the main components of the bacterial cell
membrane and the cell wall, respectively; thus, they
account for a high proportion of the total cell mass.
Despite the dense information extracted from the
NIR spectra, not all of the mid-IR peaks were suc-
cessfully correlated to the NIR spectra, and some
functional groups easily identified in the mid-IR spec-
trum are difficult to identify in the NIR spectrum.
Importantly, this paper facilitates practical use of cer-
tain theoretical conclusions and demonstrates that
transferring a high CCR of bacteria identification
from mid-IR spectroscopy to NIR spectroscopy can
be performed by correlation spectroscopy. There is
still considerable research required before regular
use of NIR spectroscopy for strains identification is
accepted within the scientific community, but we
believe that this study will help to better understand
the spectral image of some biomolecules.
Acknowledgements
The spectral data were obtained by using NIR and mid-IR
instruments located at the NIR/mid-IR Spectroscopy Unit
of the Central Service for Research Support (SCAI) of the
University of Cordoba (Spain). The preparation of this
paper was assisted by the general student development proj-
ect in progress at Brno University of Technology (Czech
Republic).
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article.
71
Funding
The author(s) disclosed receipt of the following financial
support for the research, authorship, and/or publication
of this article: The research was partially supported by
Project AGR 2012-1906 from the Andalusian Government
(Spain) and Research Group AGR-170 HIBRO.
ORCID iD
Pavel Krepelka https://orcid.org/0000-0001-8663-0481
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