Evaluation of Biological Contaminants in Foods by Hyperspectral Imaging A Review

International Journal of Food Properties
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Evaluation of biological contaminants in foods by

hyperspectral imaging: A review
Ricardo Vejarano, Raúl Siche & Wendu Tesfaye
To cite this article: Ricardo Vejarano, Raúl Siche & Wendu Tesfaye (2017) Evaluation of
biological contaminants in foods by hyperspectral imaging: A review, International Journal of
Food Properties, 20:sup2, 1264-1297, DOI: 10.1080/10942912.2017.1338729
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INTERNATIONAL JOURNAL OF FOOD PROPERTIES
2017, VOL. 20, NO. S2, S1264–S1297
https://doi.org/10.1080/10942912.2017.1338729
Evaluation of biological contaminants in foods by hyperspectral

imaging: A review
Ricardo Vejaranoa,b, Raúl Siche a
, and Wendu Tesfayec
a
Facultad de Ciencias Agropecuarias, Universidad Nacional de Trujillo (UNT), Ciudad Universitaria, Trujillo, Peru;
b
Facultad de Ingeniería, Universidad Privada del Norte (UPN), Trujillo, Peru; cDepartamento de Química y Tecnología
de Alimentos, Universidad Politécnica de Madrid (UPM), Madrid, Spain
ABSTRACT ARTICLE HISTORY

Responding to the ever-growing concern about safe foods and security, the food Received 16 January 2017
industries are forced to seek an emerging technology capable of detecting and Accepted 1 June 2017
quantifying contaminations, especially those of biological origin. Among the
KEYWORDS
different emerging technologies, hyperspectral imaging is considered a good Hyperspectral imaging;
alternative as it can be easily applied at all steps of the food production process Microbial contaminants;
and is a non-destructive technique. This paper reviews targeted analytical Toxins detection; Parasites
applications of hyperspectral imaging in monitoring biological contaminants detection; Image technology
in food. First, traditional techniques for detection of biological contaminants in
foods are presented, where disadvantages for practical applications are high-
lighted and explained in detail. Second, prominent applications of hyperspectral
imaging from the last decade to food safety and quality assessment are
reviewed, specifically focusing on both deteriorative and pathogenic microor-
ganisms, microbial toxins, and parasites; whether acting individually or collec-
tively in spoiling food products and/or represent a health risk to the consumers.
Finally, relevant current and future challenges, advantages and disadvantages of
hyperspectral imaging applications are briefly examined.
Introduction
Food quality and safety problems are receiving increasing attention in both developed and developing
countries,[1] and these issues are frequently confronted in our daily life because in today’s markets there is
an increasing demand for safe and quality products and also because food safety legislations have become
more and more stringent.[2]
The food industry is actually focused on developing innocuous products and requires a constant
commitment to the design and implementation of procedures and systems to control various para-
meters in food products (Fig. 1). Currently available analytical techniques are mostly slow methods
and destructive too. Therefore, it is urgent to develop non-invasive, efficient, and quick testing method
for monitoring food quality and safety.[3] Of all the available options, hyperspectral imaging (HSI)
technology is shown as one of the most promising alternatives, being a non-destructive analysis
technology that can easily engage in productive processes.
Several recent review articles have been published on applications of HSI which is primarily
concerned with overall food quality and safety evaluation,[3–13] and others have been mainly focused
on determination of chemical and microbiological contamination on food products.[1,2,14,15] However,
there is not any paper which specifically addresses on the application of HSI to evaluate different
contaminants of biological origin in food products, such as spoilage microorganisms that can attack
agricultural crops and cause losses in production output and quality; microorganisms causing
CONTACT Raúl Siche rsiche@unitru.edu.pe Facultad de Ciencias Agropecuarias, Universidad Nacional de Trujillo (UNT).
Av. Juan Pablo II S/N, Ciudad Universitaria, Trujillo, Peru.
© 2017 Taylor & Francis Group, LLC
INTERNATIONAL JOURNAL OF FOOD PROPERTIES S1265
Figure 1. Main parameters controlled in food production.
deterioration and quality loss in ready-to-eat foods, especially pathogens and/or their toxins; different
organic residues that can be a vehicle for different microbial contaminants; and parasites.
It is estimated that between 10% and 50% of agricultural production, especially grains and
vegetables, are lost each year as a result of microbial contamination.[16] Besides, it is estimated
that 42% of foodborne illnesses are mainly caused by contaminating microorganisms, especially
pathogenic bacteria.[17] For instance, in the United States, it is estimated that each year roughly 1 in 6
Americans (about 48 million people) gets sick, where around 128,000 were hospitalized, and 3000
died of foodborne diseases.[18] Therefore, this review focuses on the most notable application of HSI
for detecting such contaminations at any level of food production chain, which includes the
production of agricultural raw materials, and the industrial processing, packaging, and the storage
conditions, that is, from farm to table.
Traditional techniques used to detect biological contaminants in foods

Table 1 summarizes some of the most common techniques used for detecting the presence of
contaminants such as bacteria and/or their toxins, fungi and/or their toxins, viruses, and parasites in
different food products.
Evaluation of viable microorganisms

Many of the employed techniques have some disadvantages for practical applications, for example, the
culture and colony counting methods, commonly used as standard approaches for detection of
microorganisms, have major drawbacks being labor-intensive and time-consuming, and often take
2–3 days for initial results, and up to 7–10 days for confirmation.[19] On the other hand, early detection
of pathogens can be useful to prevent potential negative effects on consumers’ health and degradation
of the food products, which is not always possible by conventional plate count, especially with
pathogens such as Escherichia coli.[20]
For its part, the conventional direct microscopic techniques are time-consuming, particularly when a
large number of samples are analysed or they do not provide complete quality and safety assurance
S1266 R. VEJARANO ET AL.
Table 1. Some of most common analytical techniques for detecting biological contaminants in foods.
Analytical technique Contaminant Food Reference
Plate count Escherichia coli Fresh plant-based foods [20]
Total bacteria Beef [74,145,146]
Total bacteria Chicken meat [143]
Total bacteria Salmon and carp [144,170,178]
Total bacteria and PPC Pork [152]
LAB Salmon [172]
Pseudomonas, Brochothrix Different meats [101,141,142,180]
thermosphacta,
and Enterobacteriaceae
Microscopy Bacteria and fungi Different foods [21]
ELISA Fungal toxins Cereals [37,39]
Bacterial toxins Meat, sausage, milk, and [38,40]
mayonnaise
Virus Crops [89,90]
Anisakis simplex proteins Sea products [67,71]
Trichinella Pork [68]
Taenia saginata Beef and pork [69,70]
PCR Virus Crops [90]
Salmonella Meats and milk [197]
Bacteria Fruit crops [198]
Fungal toxins Cereals [199]
Taenia saginata Beef and pork [66,70]
HPLC and TLC Fungal toxins Cereals [41-43]
LC-MS Anisakis simplex proteins Fish [72]
Detection by fluorescence Fungal toxins Cereals [43,48-50]
Ultrasound LAB, Aspergillus niger, and Fruit juices [30]
Saccharomyces cerevisiae
Parasites Fish [65]
Vis/IR spectroscopy Microbial contamination Fresh and processed foods [34]
Fungal contamination Red pepper powder [35]
Visual inspection Parasites Fish [60,61]
MID-FTIR Trichinella Pork [193]
ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; LAB, lactic acid bacteria; LC-MS,
liquid chromatography–mass spectrometry; MID-FTIR, mid-infrared Fourier transform spectroscopy; PCR, polymerase chain
reaction; PPC, psychrotrophic plate count; TLC, thin layer chromatography.
responses,[21,22] and do not adequately differentiate between viable and non-viable cells. As alternative,
methods based on cell staining have been designed,[23,24] which in turn can be tedious and expensive.
Meanwhile, the immunology-based methods such as enzyme-linked immunosorbent assay (ELISA)
have been successfully employed for the detection of microbial contamination (Table 1). However,
with excessive total microbial count, these methods show low sensitivity, low affinity of the antibody to
the microorganism or other analysed objects, and potential interference from contaminants.[25]
Furthermore, the polymerase chain reaction (PCR) method is also a broadly used method for the
detection of pathogens in foods (Table 1). In spite of its advantages, PCR is considered to be excessively
expensive and complicated, and skilled workers are needed to carry out the tests.[19] Besides, PCR and
ELISA tests are destructive and take longer to get results, as well as have limited application at
laboratory level,[26] and there could be possible interference of inhibitors present in foods,[27] which
could produce erroneous results with these techniques.
Other alternatives include the use of biofunctional magnetic nanoparticles (BMNPs) combined
with ATP bioluminescence, for example, for rapid detection of E. coli in pasteurized milk;[28] as well as
a quartz crystal microbalance (QCM)–based DNA biosensor to detect the main viral RNA encoding G
protein in viral haemorrhagic septicemia infection (VHS),[29] one of the most serious viral diseases
damaging both fresh and marine fish species. According to the obtained results, these detection
techniques are rapid, specific, and sensitive. However, most of them are technically complicated and
costly, and require well-trained specialists.[20]
Ultrasound technology is also used in food-quality characterization, which is based on the variation of
flight time of the ultrasonic waves as a result of chemical changes induced by microbial contaminants.[30]
However, among its limitations the difficulty to be applied for a wide variety of microorganisms with
different replication time is highlighted. This technology usually has been applied for detecting seal
defects in food packages, for predicting chemical composition, and for detecting physicochemical
changes and foreign objects, such as bone, glass, or metal fragments.[4]
Regarding visual inspection, it is difficult for the workers to detect contaminated products,
especially when they work on an inspection table, examining foods moving at a relatively high
speed.[31] Also, the identification of microbial contamination by imaging appears to be a good
alternative; nevertheless, conventional imaging lacks the ability to measure material composition
through spectral analysis.[32] Besides, operating at visible wavelengths (i.e., monochromatic or RGB
images), it is incapable in differentiating samples with similar colour, classifying complex objectives,
predicting chemical components, and in detecting invisible defects.[11] Likewise, regarding detection of
fecal residue and/or digested materials, the problem associated with the classification accuracy is that
only the thick feces could be observed and easily identified, while the thin layers normally become
problematic for detection when only one band is used.[33]
For quantitative analysis, visible (Vis) and near-infrared (NIR) spectroscopies[34,35] are not indepen-
dent of the disadvantages arising from the reference method used for calibration, which requires a series
of samples with known analyte (standard) concentrations.[14] For instance, information about “where”
and “how” microorganisms are physically distributed in food samples cannot be acquired, which is
essential for visualizing the distribution of the microbial spoilage, because measurements only involve a
limited portion of the sample.[7,36] Moreover, the spectroscopy is applicable to characterize homogenous
samples by providing mean values of analysed samples, so a single measurement may be misleading to
represent the bulk value of the entire sample when heterogeneous samples are evaluated.[7]
As a solution to this situation, HSI combines the complementary approaches of spectral analysis
and image processing, thus offering the advantage to evaluate simultaneously the composition
(spectral information) and the external characteristics (traditional image analysis) in different
foodstuffs.[3,6]
Evaluation of microbial toxins

Different techniques have been explored to determine the presence of microbial toxins, such as
ELISA,[37–40] high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC),
gas chromatography (GC),[41–43] molecular identification techniques,[44] and other techniques based
on chemical analysis. Although these techniques have many merits such as specificity, accuracy,
selectivity, and very low limit of detection, most of them are generally expensive, labor-intensive,
destructive, and time-consuming to achieve appropriate results,[45] some of them are unable to handle
a large number of samples,[11] require unfriendly organic reagents,[46] and their application is also
limited at a laboratory level. In addition, there is risk of discarding an entire lot or accepting a
contaminated lot as safe due to the uneven distribution of contaminated products in storage[47] with
the economic losses that it implies.
Detection of microbial toxins by fluorescence (induced reaction between peroxidase enzyme with
kojic acid and toxins) is also used because certain photosensitive compounds fluoresce when they are
exposed to shortwave radiation such as UV radiation,[43,48–50] for example, toxins such as aflatoxins
(produced by Aspergillus flavus and Aspergillus parasiticus), while others, such as fumonisin, cannot
be detected by fluorescence. Besides, in some cases A. flavus has also been noted to produce kojic
acid, which can convert to fluorescent compounds, but does not produce aflatoxin,[51] likely to give
false positive or false negative results, and therefore discarding an entire lot or accepting a con-
taminated lot as safe.
Likewise, transient infrared spectroscopy (TIRS) is a technique for online, non-contact detection of
A. flavus, which is potentially effective in screening bulk quantities of grain,[52] for example, early tests
based on TIRS spectral differences to distinguish healthy from infected grains. However, the limitation
of TIRS is the special requirements on heating the media surface for producing proper thermal
emission,[53] which is inviable in large batches of food products.
Techniques for detection of insect infestation

There is an increasing need to detect insect infestation during postharvest handling practices and before
the food products are shipped to the market. There is not extensive information available in the
bibliography regarding studies performed on the possible methods for identifying the insect infestation
in agricultural products,[54] although several technologies such as acoustic devices and signal processing
methods, conductivity, or NIR spectroscopy have been studied. However, these technologies are complex
and destructive, and detecting dead and larva insects is difficult through these.[55]
Likewise, X-ray technique has also been researched for insect detection,[56,57] but this technology is
not currently in use because of its cost and difficulty in effectively discriminating normal and infested
tissues.[58] On the other hand, since the insect larvae usually produce small holes on the surface of food
products, the common machine vision technology in the visible range can be a possible means of
recognition. Nevertheless, penetrating the inner part of the samples using machine vision technology
with a visible light source is hardly possible.[55] Besides, this technology is sensitive to surface features
and may provide information regarding other types of surface damage with similar symptoms to holes
as in the case of insect larvae. Therefore, machine vision has been considered to be unreliable for
inspecting the insect infestation in agricultural products.[57,59]
Evaluation of contamination by parasites

Fish fillets are inspected by transillumination on candling tables and parasites are removed manually
by trained personnel,[60] achieving to identify up to 70% of contaminated pieces.[61] However, this
procedure is expensive; the results depend on the training and skills of inspectors and are prone to both
human error and inspector-to-inspector variation.[60,61] Besides that, it is performed at room tem-
perature, which increases the risk of degradation by endogenous enzymes and contaminant
microorganisms.[61,62]
Other techniques explored include the use of fluorescence to spot parasites.[63] However, this
method was limited to the detection of surface nematodes, because the UV light does not penetrate
deeply into the fish muscle, therefore is no longer commercially available.[64] Hafsteinsson et al.[65]
reported detection of parasites deeply embedded into the fish muscle by use of ultrasonic waves,
on the basis of the difference in acoustic properties between the fish tissue and parasite. The
drawback of this method was the requirement of direct coupling between detector and measure-
ment medium, therefore it is one of the reasons why it has not been industrialized.
Also, PCR and ELISA have been successfully applied for the detection of parasites in
meat,[66–71] although with the same disadvantages as previously mentioned. For its part, liquid
chromatography–mass spectrometry (LC-MS) has been shown to be a powerful tool for
isolating proteins from Anisakis.[72] Although chromatographic techniques have many merits
such as specificity, accuracy, selectivity, and very low limit of detection, most of them are
generally expensive, labor-intensive, destructive, require long time to get the results and
introduce unfriendly chemicals,[45,46] and their application is also limited to a laboratory level.
Because of these disadvantages, the traditional analytical techniques are normally not suitable for online
and real-time detection, and for large-scale operations in a rapid and non-destructive/non-invasive way.[5,14]
So that, of all the available options, HSI technology is shown as one of the most promising alternatives,
which overcomes all of the aforementioned disadvantages of conventional technologies used to evaluate
foodstuffs, because these methods are not practical when fast analysis and early detection of biological
contaminants in industrial and commercial processing are required with the minimum human
intervention.[6]
Hyperspectral imaging technology

Also known as “imaging spectroscopy” or “imaging spectrometry,” HSI is an emergent technology that
integrates the advantages of spectroscopy and imaging, allowing to evaluate simultaneously the
composition (spectroscopic component) and external characteristics (traditional image analysis) of a
sample.[3,6,8,11] That is, the spectroscopy responds to the questions “what” and “how much” and the
image analysis provides an answer to the question “where.” Therefore, HSI systems have had a
significant influence on the outcome to the combined questions “where and how much of what,”
providing a comprehensive characterization of a sample.[73]
Figure 2a. shows a typical laboratory experimental setup of the HSI system. The hyperspectral
images obtained are three-dimensional data cubes (hypercubes) made up of hundreds of images of
the same object at different spectral wavelengths (Fig. 2b), where spectra of each pixel can be used to
characterize the composition of that specific position, and surface-feature information can be
obtained according to the spatial images.[4,11]
The basic principle of HSI is based on the fact that all materials reflect, scatter, or absorb energy
differently when they are subjected to an electromagnetic radiation source at different wavelength ranges
due to the difference in their chemical composition and physical structure.[7] Light scattering is related
with physical characteristics of the samples such as particle size, cellular structure, and tissue density;
whereas light absorption is related with chemical composition of the objective material.[74,75]
Each food constituent has a typical “spectral signature” or “spectral fingerprint” when it interacts
with the incident light, which can be used to characterize, identify, and discriminate between different
samples.[76] This “spectral signature” tells about its chemical composition and can be plotted against
different wavelengths, resulting in the feature curve of reflectance, absorbance, or transmittance for
each material (Fig. 2c).
Although the constituents of a sample present certain reflectance (or absorbance) values at specific
wavelengths, as in the case of conventional spectrophotometers, it is possible to obtain complete
Figure 2. Schematic of the hyperspectral imaging (HSI) system: (a) acquisition of hyperspectral image; (b) hypercube: spectral and
special information in a hyperspectral image at different wavelengths; (c) spectral signature for each constituent of the sample
plotted against different wavelengths.
information of such constituents in different spectral bands, from ultraviolet–visible (UV–vis) to

NIR,[45] which allows to measure a wide range of quality parameters, as well as the presence of
contaminants and their spatial distribution in a given food sample.[77]
There are several studies regarding food analysis by HSI, which offers advantages like speed,
accuracy, reliability, and furthermore being a non-destructive analysis technology that can be applied
along the different production phases, simultaneous assessment, and real-time data processing of
numerous sample chemical and physical properties,[7,53] in addition to the simplicity in sample
preparation, and being a chemical-free analysis technology,[15] aspects that with other technologies
are not always possible.
Since 2011 literature on the use of HSI in food analysis has increased considerably,[78] indicating
that the acceptance of this technology is relevant because of the advantages mentioned previously.
These studies deal with various parameters ranging from chemical composition, nutritional value,
sensory attributes, and detecting defects and contaminants in different foodstuffs (details are given in
Fig. 1). Among an increasing number of food contaminants, those of biological origin may cause a
marked accelerated chemical and structural degradation within a short period of time. These changes
could be detected depending on the magnitude of scattering or absorption intensity of the incident
radiation by the sample during the analysis.[74,75,79]
In this sense, this review is focused on the most relevant studies conducted using HSI as an alternative
tool and summarizes the outstanding results in the detection and evaluation of contaminants of
biological origin such as pathogenic microorganisms, undesirable and/or harmful substances as a result
of microbial metabolism (such as toxins), as well as the presence of viruses, parasites, and organic
residues that can act as carriers of contaminating organisms; which acting individually or collectively can
deteriorate food products and/or represent a health risk to consumers.
Applications to evaluate biological contaminants in plant-based foods

Microbial contamination in agricultural crops
Early detection of crop diseases benefit farmers as they can remove infected plants quickly before
diseases spread and inflict further damage.[80] In this sense, adequate monitoring of cultivars can be a
useful and cost-effective approach for mapping of infected crops, for instance, by the using hyper-
spectral airborne imaging. In Table 2, a summary of studies related to spectral imaging for detecting
microbial contamination in different cultivars is presented.
A pioneering study was done by Ausmus and Hilty,[81] where spectral reflectance (400–2700 nm) was
used to evaluate the presence of fungi Helminthosporium maydis and of the maize dwarf mosaic virus
(MDMV), by taking into account the injuries and colour changes in the maize leaves. Their results revealed
that healthy, MDMV infected, and H. maydis–infected leaves could be spectrally differentiated in the NIR
range (800–2600 nm), especially at early infection stages when symptoms are not yet visible by direct
observation (reflectivity: healthy leaves > MDMV-infected leaves > H. maydis–infected leaves).
Later, Hamid Muhammed and Larsolle[82] used HSI (360–900 nm in reflectance mode) to detect the
fungal disease known as “tan spot” in wheat, caused by the pathogenic fungi Drechslera tritici-repenti.
However, according to the authors, the possibilities for fungal disease control using HSI has to be
further investigated.
On the other hand, contamination by Fusarium ssp. is a serious problem for the cereal industry due
to its potential capacity of producing mycotoxins. Detection of contamination at the early stages can be
an effective strategy to prevent substantial loss in cereal production as well as prevent that toxins reach
the consumer in later stages of the production chain. In this regard, Delwiche and Kim[83] used HSI
(425–860 nm) to identify wheat grains affected by “Fusarium head blight”, disease that causes wheat
grain to be shriveled, underweight, difficult to mill, and is a health concern because of the possible
production of the mycotoxin deoxynivalenol. According to the authors, this disease is more suitable to
detect in the NIR region than in the visible spectrum.
Table 2. Spectral imaging applications for detecting microbial contamination in agricultural crops.
Cultivar Contaminating agent Aim Wavelength (λ, nm) Reference
Maize ● Fungi: Spectral differentiation in the NIR region 400–2700 [81]
Helminthosporium (800–2600 nm) between healthy, MDMV
maydis infected, and H. maydis–infected leaves
● Virus: MDMV
Wheat Fungi: Fusarium Correct classification between diseased and 400–1000 [84]
culmorum healthy ear tissues
Strawberry Fungi: Colletotrichum Early detection of infection stages of 400–1000 [80]
gloeosporioides strawberry foliar anthracnose
Citrus fruit Bacteria: Xanthomonas Multispectral algorithms to differentiate 450–930 [85]
axonopodis “citrus canker” from other peel conditions
Grapevine Virus: GLRaV-3 Evaluation of the reliability of hyperspectral 400–1000 [92]
airborne imaging to remotely detect GLRaV-3
vineyards
Correct classification between infected and 350–2500 [93]
healthy vine leaves
Watermelon seeds Virus: CGMMV To discriminate virus-infected watermelon 946–2016 [94]
seeds from healthy ones
MDMV, maize dwarf mosaic virus; GLRaV-3, grapevine leafroll-associated virus-3; CGMMV, cucumber green mottle mosaic virus.
Also, Bauriegel et al.[84] evaluated wheat plants using HSI (400–1000 nm) in order to detect “Fusarium
head blight” disease before harvest. In Fusarium-infected ear tissues, absorption in the range of the
chlorophyll bands (560–675 and 682–733 nm) rapidly decreased with development of infection as a
result of destruction of chloroplasts. Likewise, they applied spectral angle mapper (SAM), achieving 91%
of correct classification for the degree of disease. Nevertheless, due to the time-consuming and complex
classification algorithm (SAM), this method is not recommended for practical applications. So a fastest
classification was achieved with a new head blight index (HBI), based on principal component analysis
(PCA) to identify distinct wavelength ranges with pronounced difference between healthy and diseased
tissues, obtaining an accuracy rate of 67%, which could be improved with this index.
Regarding to fruit crops, Qin et al.[85] applied HSI (reflectance mode at 450–930 nm) for early
detection of “citrus canker”, a bacterial disease caused by Xanthomonas axonopodis, where symptoms
are visible just after 7–10 days of infection. Correlation analysis (CA) and PCA were used for
hyperspectral band selection. Based on a two-band ratio selected by CA (R834/R729), algorithms for
multispectral image were developed to differentiate canker from other peel conditions, obtaining an
overall classification accuracy of 95.7% highlighting that the two-band ratio images have great
potential to be adopted by a multispectral imaging (MI) system for early and real-time “citrus canker”
detection.
The same two-band ratio (R834/R729) selected by CA gave a correlation value of 0.811 and
classification accuracies between 93.3% and 96.7% in a previous study carried out by Zhao et al.[86]
Later, on the basis of these key wavelengths, Qin et al.[87] developed an online commercial fruit sorting
machine that achieved an overall classification accuracy of 95.3%, operating at speed of 5 fruits/second.
Similarly, early detection of infected vine by grapevine leafroll-associated virus 3 (GLRaV-3), an
important tool to overcome a decrease in grape production yield,[88] is an aspect that with conventional
techniques such as ELISA and PCR[89,90] would not be possible. This virus reduces the total level of
chlorophyll in vine leaves, which can be detected by measuring the differences in reflectance between
infected leaves and healthy ones.[91] Besides, although there is no specific treatment for the disease,
early detection can limit its spread.[92]
According to Naidu et al.,[93] the presence of this virus in vineyards of Cabernet Sauvignon and
Merlot tends to decrease the absorption of chlorophyll at 550 nm in infected leaves (due to changes in
the pigment), but without any visible symptoms. They applied a stepwise discrimination analysis
(SDA) to select key wavelengths that discriminate infected from healthy vine leaves in the spectral
range between 350 and 2500 nm, obtaining an overall accuracy of 0.81. More recently, MacDonald
et al.[92] have showed that remote monitoring (hyperspectral airborne imaging at 400–1000 nm) of
GLRaV-3 infected Cabernet Sauvignon vineyards can be a useful and cost-effective approach for
mapping infected crops, with a success rate of leafroll detection of 94.1% using a classification and
regression tree (CART) algorithm. However, according to the authors, future studies should focus on
the use of this technology for detecting GLRaV-3 in other grape varieties, as well as other grapevine
pathogens.
Similarly, early detection of “anthracnose” would avoid a decrease in the strawberry yield because not
only the healthy and symptomatic stages can be recognized but also at the incubation stage before any
“anthracnose” symptoms become visible. A pioneering study has been developed by Yeh et al.,[80] who
have demonstrated that the different infection stages can be identified through HSI (400–1000 nm) in
artificially inoculated strawberry leaves with Colletotrichum gloeosporioides to simulate foliar “anthrac-
nose”. Different statistical analysis methods were evaluated, with the results indicating SDA as the most
appropriate method, with an average accuracy between 80% and 93%.
Finally, the cucurbit diseases caused by cucumber green mottle mosaic virus (CGMMV) have led to a
serious problem to cucurbit producers because pathogen-infected seeds transmission phenomenon is
difficult to combat. Recently Lee et al.[94] have applied an NIR–HSI system (reflectance mode at
946–2016 nm) to discriminate virus-infected watermelon seeds from healthy ones using key wavelengths:
1411, 1456, 1792, and 1867 nm, selected by partial least square discriminant analysis (PLS-DA), obtaining
a classification accuracy of 83.3%. Besides that, the authors propose phenolic compounds as a prominent
key discriminating factor between virus-infected and healthy seeds where the most common feature of
major peaks were consistent with the absorption peaks of these compounds, which plants produce to
defend themselves not only against herbivores, but also against competing plants and microorganisms.
These aforementioned results clearly demonstrate that the use of spectral methods provide useful
information to avoid significant agricultural productivity losses, because it would be possible to
detect infected cultivars before the damage becomes visible.[26,80] Additionally, compared with
traditional detection methods, HSI offers a potentially valuable alternative for monitoring crop
diseases, since this technology is cost-effective, reliable, and automatable.[92]
Contamination by microbial toxins in plant-based foods

Microbial toxins are among the most controlled food contaminants and its consumption can lead to health
problems such as jaundice, liver carcinomas, esophageal cancer, neural tube defects, immunosuppression,
etc.[95,96] These substances are metabolites produced by fungi (mycotoxins) and bacteria (bacterial toxins),
and the most common in food products are aflatoxins produced by A. flavus and A. parasiticus,[97]
ochratoxin produced by Aspergillus,[96,98] fumonisins and trichothecenes produced by Fusarium,[96] and
patulin produced by Penicillium, Aspergillus, or Byssochlamys.[99]
Analytical techniques such as ELISA, HPLC, TLC, GC, and visual analytical techniques such as
fluorescence-based detection methods[43,45,48–50] are applied for detecting toxins in food products.
However, these techniques are susceptible of the disadvantages mentioned earlier. There is thus a
growing need for rapid, non-destructive, non-invasive, and accurate techniques that do not require a
major resource investment. The HSI technology is the best option to fulfill these requirements,
allowing not only the detection of microbial toxins in foods[45,100] but also the detection of the presence
of viable microorganisms, and either toxigenic or non-toxigenic strains from both bacteria[20,101] and
fungi.[22,43] Table 3 summarizes some of the research works carried out using HSI technology to
evaluate contamination caused by viable microorganisms and/or their toxins.
Pearson et al.[102] evaluated the presence of aflatoxin in maize grains by using of HSI (500–950 nm)
and by an affinity chromatography commercial procedure. Results were similar when using either
discriminant analysis (DA) or partial least squares regression (PLSR) for classification, and more than
95% of the grains were correctly classified as containing either high (>100 parts per billion, ppb) or low
(<10 ppb) levels of aflatoxin. Besides that, they determined that the wavelength of 735 nm is related to the
presence of this toxin. Also, Yao et al.[103] estimated aflatoxin concentration in maize grains inoculated
with A. flavus spores, obtaining a R2 of 0.72 using a multiple linear regression (MLR) model. The
Table 3. Hyperspectral imaging (HSI) for detecting microbial contamination and damages caused by insects and/or their organic
residues in plant-based foods.
Contaminant Food Modea Wavelength (λ, nm) Accuracy References
Microbial toxins
Aflatoxin Maize F 450–500 94.4% [43]
Maize R 1100–1700 96.9% [48]
Maize R 400–1000 98% [106]
Maize R 550–1700 95% [102]
Maize T 500–950 95% [102]
Fumonisin Milled maize R 720–940 R2 = 0.68 [42]
Ochratoxin A Barley R 1000–1600 100% [98]
Viable microorganisms and diseases
Aspergillus and Fusarium Maize R 400–1000 97.0%, 94.5% [45]
Aspergillus and Penicillium verrucosum Barley R 1000–1600 100% [98]
Fusarium Maize A 1000–2498 R2 = 0.98 [100]
Aspergillus niger Wheat R 1000–1600 92.9% [112]
Aspergillus glaucus Wheat R 1000–1600 87.2% [112]
Penicillium Wheat R 1000–1600 99.3% [112]
Aspergillus oryzae Rice R 400–1000 R2 = 0.97 [118]
Decay Citrus fruit R 325–1100 98.6% [130]
R 460–1020 87.5–95.5% [131]
Canker Citrus fruit R 450–930 95.7% [85]
R 450–930 93.3–96.7% [86]
R 730 and 830 95.3% [87]
Fecal contamination Apple F and R 400–1000 100% and 99.5% [125]
Apple F 480–780 99% [127]
Escherichia coli Spinach R 400–1000 R2 = 0.97 [20]
Frass Tomatoes F 400–800 >99% [138]
Damaged caused by insects
Fruit fly Cucumber T and R 450–1000 82–93% [58]
Insect infestation Cherries T and R 580–1550 [59]
Jujube R 900–1700 93.1% [134]
Callosobruchus maculatus Mung bean R 1000–1600 82–85% [200]
Etiella zinckenella Treitschke Soybean T 400–1000 95.6% [55]
R2, coefficient of determination.
a
A, absorbance; F, fluorescence; R, reflectance; T, transmittance.
multivariate analysis of variance (α = 0.01) showed significant differences between four aflatoxin groups
tested: <1, 1–20, 20–100, and ≥100 ng/g ppb, and the classification accuracy ranging from 0.84 to 0.91
when a threshold of either 20 or 100 ng/g was used.
Later, Yao et al.[43] inoculated maize ears with two strains of A. flavus (one toxigenic strain and
other non-toxigenic strain), and production of aflatoxin in both germ and endosperm fractions of
grains was detected by an affinity chromatography commercial procedure and by fluorescence HSI
(400–700 nm), thereafter the range between 450 and 500 nm was figured out as the best spectral
region to detect fungal contamination. Results from the DA classification indicated overall higher
classification accuracy for a 100 ppb threshold on the germ side (94.4%).
More recently, Wang et al.[104] assessed the potential of NIR-HSI (1000–2500 nm) to detect
aflatoxin on maize grains, and a minimum classification accuracy of 88% was achieved and as low as
10 ppb of aflatoxin was detected by applying PCA and factorial discriminant analysis (FDA)
methods. Likewise, the same group[105] evaluated the feasibility of detecting aflatoxin in artificially
inoculated maize ears, and two wavelengths (1729 and 2344 nm) were identified to characterize
aflatoxin exclusively, achieving a higher detection accuracy (92.3%).
Moreover, applying Vis/NIR-HSI (600–1000 nm) and HPLC analysis, Wang et al.[106] analysed maize
grains contaminated with aflatoxin, and an overall classification accuracy of 98% was achieved. A
combination of PCA and FDA (PCA-FDA) was performed to detect and differentiate different levels
of aflatoxin, pointing out the detection of this toxin at 735.2 nm as it was obtained by Pearson et al.[102]
Similar results were obtained by Kandpal et al.,[48] who applied a SWIR-HSI system (reflectance mode at
1100–1700 nm) to detect aflatoxin contamination on maize grains inoculated with four different
aflatoxin concentrations: 10, 100, 500, and 1000 mg/kg. They developed a PLS-DA model to categorize
infected grains, obtaining an overall classification accuracy of 96.9%.
In the case of other food products, Kalkan et al.[47] detected the presence of aflatoxins by the
using of multispectral images and liquid chromatography (LC) in artificially contaminated hazelnut
samples with A. parasiticus. A two-dimensional local discriminant bases (LDB) algorithm was
developed and a classification accuracy of 92.3% was achieved for aflatoxin-contaminated and
uncontaminated hazelnuts. The algorithm was also used to classify fungal contaminated and
uncontaminated samples, and an accuracy of 95.6% was achieved.
On the other hand, unlike aflatoxins that can be detected by fluorescence,[43,50] fumonisin is known
for its difficulty to be detected directly by optical methods. In this respect, reflectance analyses in the
NIR region can be useful to detect this toxin in cereals. Firrao et al.[42] evaluated the presence of
fumonisin in milled maize using HPLC and multispectral images (720–940 nm), with a coefficient of
determination R2P of 0.68 for fumonisin concentration (linear regression model fitting). Furthermore,
on the basis of the predictions provided by a neural network (NN), the samples were assigned to one of
three classes named high (>2.8 ppm), medium (0.5–2.8 ppm), and low (≤0.5 ppm), for their predicted
fumonisin content.
For its part, the presence of ochratoxin A in stored barley has been recently evaluated by
Senthilkumar et al.[98] using NIR-HSI (reflectance mode at 1000–1600 nm). They have applied PCA
to select key wavelengths: 1260, 1310, and 1360 nm (to evaluate Aspergillus glaucus, Penicillium spp.,
and non-ochratoxin A producing Penicillium verrucosum–infected grains), and 1310, 1360, and 1480
nm (to differentiate ochratoxin A contaminated grains). Selected wavelengths were used as input for
statistical classifiers, which differentiated fungal infected grains with more than 80% (initial periods of
infection) and 100% classification accuracy (four weeks after infection). These authors also highlight
the fact that the time taken for image acquisition, processing, and data analysis for five grains is less
than 2 min, which would be reduced to less than 1 min when it is performed later with selected key
wavelengths.
Contamination by viable microorganisms in plant-based foods

Safety problems as a consequence of contaminants of biological origin of most plant-based foods
might be severe because a small portion of infected products may contaminate the whole batches,
and ingestion of contaminated products might incur health problems to consumers.[9] Therefore, it
is crucial and necessary to apply rapid, accurate, and non-invasive technique in food analysis
procedures to overcome previously mentioned drawbacks. In Table 3 a summarized list of
approaches to evaluate the presence of viable microorganisms and organic residues (vehicle for
transmitting microorganisms to foods) of insects and other animals is shown.
Viable microorganisms in cereals

One of the biggest problems for the cereal industry is the significant loss caused by so-called storage
fungi. If it is not eliminated by proper drying processes after harvest, it may flourish during storage
under favourable conditions,[107,108] affecting important features such as appearance, chemical
composition, or germination capacity. Apart from that, this fungal growth promotes some undesir-
able side-effects such as production of toxins and off-odours originating from the synthesis of
volatile compounds such as 3-methyl-1-butanol, 1-octanol, and 3-octanone.[109]
The possibility of spectral information for detecting characteristic compounds at certain wavelengths
is reported by Fernández-Ibáñez et al.,[41] who related the spectral range of 870–1200 nm with com-
pounds that include the functional group –NH, like amino acids and aromatic rings of furans and
phenols, formed as a consequence of fungal degradation of cereals.
HSI is one of the best alternatives for the identification of different fungal species, because it shows a
specific “spectral signature” for each species.[110] The “spectral signature” can be useful for detecting
fungal contamination in foods and identify unknown fungal species on the basis of their spectral profile
in comparison to the referential spectra of known fungal species.[45] The studies mentioned below
demonstrate the advantage of the HSI detection on microbial contamination when symptoms are not
yet visible by direct observation.
Shahin and Symons[32] designed a low-cost MI system from an HSI system (400–1000 nm), and
reported that 917 nm is a relevant wavelength to detect mildew damage on wheat grains. A partial least
square (PLS) model analysis based on four wavelengths (450, 561, 861, and 917 nm) achieved values of
R2 = 0.89 and RMSE = 0.68 (calibration model), and R2 = 0.87 and RMSE = 0.74 (validation model).
According to this study, compared with the full-wavelength models (HSI systems), the simplified
models (MI systems) present small differences in R2 and RMSE values, and the proposed MI system
could be more suitable for online applications, due to its relatively low instrument cost and high
analytical speed.[4–6,9]
Meanwhile, Singh et al.[111] used SW-NIR-HSI (700–1100 nm) for fungal detection in wheat
grains infected by Penicillium spp., A. glaucus, and Aspergillus niger. Linear discriminant analysis
(LDA) classifier was applied and classification accuracy between 97.3% and 100.0% was achieved for
healthy and fungal-infected samples.
On the other hand, it is very well known that toxigenic fungi grown in grain are toxic for humans and
animals. Jin et al.[53] used HSI (400–1000 nm) to discriminate toxigenic and non-toxigenic A. flavus
strains. The prominent dimensionality reduction technique PCA and a support vector machine (SVM)
model were successfully applied for the classification. Under halogen light source, 83% of toxigenic
strains and 74% of non-toxigenic strains were classified correctly; whereas using UV light, the classifica-
tion was 67% and 85%, respectively. Similarly, Zhang et al.[112] applied NIR-HSI (1000–1600 nm) to
evaluate wheat grain infection by different fungi. The dimensionality was reduced by PCA and a
multiclass SVM with kernel of radial basis function was used for classification, achieving differentiation
rates of 92.9% for those contaminated by A. niger, 87.2% by A. glaucus, and 99.3% by Penicillium strains.
Similarly another study realized by Del Fiore et al.[45] evaluated the presence of A. flavus, A. niger, A.
parasiticus, and Fusarium verticillioides in maize grains by HSI (400–1000 nm). PCA was performed on
the whole set of spectral data, and the principal components obtained were used to create a model for
fungal identification by DA. Results revealed that between 500 and 700 nm, the absorbance increased
while fungal and mycotoxin contamination increased, however, between 850 and 950 nm the absorbance
values decreased, especially with an A. niger strain.
The variation of absorbance observed in the visible spectrum (500–700 nm) could be caused by
fungi, which alters the spectral properties of maize grains related with colour changes,[41] while the
variations in the NIR region (850–950 nm) have been possibly due to scattering of light caused by the
increased porosity in the endosperm, as a result of fungal growth.[113]
Regarding the chemical composition changes, Farag et al.[114] observed an increase of 0.5% in the lipid
content and between 3% and 9% in the protein content, as well as an increase in the reducing sugars
content up to six fold in wheat grains, especially those infected by A. niger. These chemical composition
changes would be the reason for the altered spectral properties in contaminated grains[45] as consequence
of metabolism of these constituents by fungus. Similar results were obtained by Williams et al.[100] in
maize grains inoculated with spores of F. verticillioides and analysed using HSI (1000–2498 nm). They
observed changes in the composition of the grain components, especially with prominent peaks at 1405
and 2136 nm, which are related to the absorbance for starch (O–H stretch) and protein (N–H stretch and
a C=O stretch), respectively, substances used by the fungus for growth.[115]
For its part, Shahin and Symons[116] used Vis/NIR-HSI (400–1000 nm) to detect Fusarium-damaged
grains of wheat. LDA was applied and an overall classification accuracy of ≥92% was obtained. Later, the
same authors[117] applied PLS-DA for detecting Fusarium, however a lower overall accuracy was
achieved (90%).
More recently, Siripatrawan and Makino[118] used HSI (400–1000 nm) to evaluate rice grains
inoculated with a non-toxigenic strain of Aspergillus oryzae. Partial least squares regression (PLSR)
was used to predict fungal growth from the HSI reflectance spectra, obtaining a high R2 (0.97) between
formed colonies and spectral data. Besides this, they also observed that spectral reflectance is inversely
proportional to the infection level, which would be related to the formation of cellular structures of the
fungus (as spores and mycelium), and synthesis of metabolites and changes in the chemical composition
and colour of the grains.[41,45,113] Also, higher fungal growth was observed in the portion of germ, which
is the fraction rich in proteins, lipids, and minerals, while the endosperm is mainly constituted by
starch.[119]
Viable microorganisms in fruits and vegetables

Fruit and vegetables are exposed to different sources of microbial contamination. At the early
growing stage, the most common sources are organic residues from soil and water used for
irrigation. Other vehicles are mainly insects that can spread pathogens through their bite or by the
means of their organic residual deposits on and around plants, while in postharvest stages the main
source is the cross-contamination.
Bacterial contamination. In recent years, diseases caused by foodborne pathogens such as E. coli,
Salmonella, Listeria monocytogenes, or Shigella flexneri have become an important public health
problem in the world.[120–123] Irrigation-induced contamination is one of the principal routes of
vegetable spoilage, especially in those crops where the edible portion is in contact with the soil, such
as lettuce, spinach, etc., which may be easily contaminated by pathogenic bacteria. Therefore, early
detection plays a crucial role in protecting the consumer against infectious microorganisms, so being
that the HSI technology one of the best options in this regard.
E. coli is among the most common foodborne pathogens, naturally present in the intestinal flora
and whose presence is used as an indicator of food fecal contamination. Siripatrawan et al.[20] evaluated
the presence of E. coli after inoculating it into spinach leaves and analysed by HSI (400–1000 nm),
obtaining a coefficient of determination R2 of 0.97, between plate count method and the spectral
reflectance computed using PCA (to remove redundant information of the hyperspectral data). An
artificial neural network (ANN) algorithm was used to construct a prediction map of all pixel spectra of
an image by colouring each pixel with respect to the calculated number of E. coli, expressed as log
(CFU/g). These results suggested that tandem HSI-chemometrics can be useful for a rapid and
innovative approach, where an early detection of pathogenic bacteria in packaged fresh foods is
required.
Furthermore, these pathogenic bacteria are closely associated with fecal matter as in the case of
proximity of food crops and processing operations to livestock or wildlife intrusion.[124] Comparisons
between HSI reflectance and fluorescence modes for detecting fecal contamination was performed by
Kim et al.[125] They used a commercial machine to sorting artificially contaminated apples at speeds of
three and even more samples per second, achieving an accuracy of 100% by using HSI in fluorescence
mode. For reflectance mode, the classification accuracy was 99.5%.
Yang et al.[126] employed an HSI system (fluorescence mode at 320–400 nm) for the detection of
bovine fecal contaminants on lettuce and spinach leaves, and a two-band ratio, 666 nm/680 nm, to
differentiate the contaminated spots from uncontaminated leaf area was used. Later, the same research
team[127] developed and evaluated three multispectral algorithms derived from hyperspectral line-scan
fluorescence imaging (481–780 nm) for the detection of fecal contamination on apples at four
wavebands: 680, 684, 720, and 780 nm, and using linear regression models they detected 99% of
fecal contamination spots on apple surfaces. The study highlighted that this fast and non-destructive
method for detection of fecal contamination can be implemented in the food production chain to help
the farmer and manufacturer to prevent or minimize the potential foodborne illness.
Fungal contamination. This is a serious problem since a small set of infected fruit or vegetables can
contaminate the whole batch, especially during the storage and transportation processes. Diseases such
as “green mold” (caused by Penicillium digitatum) and “blue mold” (caused by Penicillium italicum)
are two examples, which affect several cultivars over the whole world.[128] Fungal infection at early
stages cannot be detected with the naked eye because in the most of cases the appearance of the
damaged fruit is very similar to the uncontaminated ones,[31] and therefore cannot be detected in
postharvest by manual inspection.
Mehl et al.[129] used HSI (430–900 nm) to analyse apples contaminated by fungi. An asymmetric
second difference method using a chlorophyll absorption band (685 nm) and two bands in the NIR
region (722 and 869 nm) provided an excellent detection of the defective/contaminated portions of the
apples examined. Also, the carotenoid absorption band (450 nm) offered good contrast between whole-
some apples with respect to samples affected by fungal contamination, either soil contamination or
bruises. Besides that, this study showed that the asymmetric second difference and PCA methods give
very similar results. However, PCA is complex to use, because it requires longer data processing time,
while the asymmetric second difference method requires only three wavelengths and much less compu-
tation time to process the images. So, it can be easily implemented in a three-band MI system.
Citrus fruit also are susceptible to fungal contamination. These fruits are manually selected by trained
personnel exposed to a dangerous UV light source;[12,130] furthermore, it is not always possible to detect
the presence of fungi in the early stages of infection. Among fungal species that cause spoilage in citrus
fruit are included P. digitatum and P. italicum, which are also analysed by HSI.[131]
In this regard, an HSI system (320–1100 nm) was employed for early detection of rottenness caused by
P. digitatum in mandarins.[132] The optimal number of bands required to optimize the classification was
estimated to be 20, selected by genetic algorithms based on LDA (GA-LDA). The classification rate of
contaminated samples, obtained by CART, was above 91%, highlighting the capability of HSI for early
detection of this kind of contamination, which is extremely relevant from the economical point of view.
More recently, Folch-Fortuny et al.[31] have developed a N-way partial least squares regression
discriminant analysis (NPLS-DA) method to detect symptoms of P. digitatum in citrus fruits using
Vis/NIR-HSI (650–1080 nm). They selected five key wavelengths: 650, 660, 700, 750, and 760 nm to
discriminate between control and contaminated oranges, where 96.8% of control samples and 95.7% of
fungus-contaminated fruits were correctly classified. In accordance with the results obtained and from
a practical point of view, the NPLS-DA model was an effective means to reduce the losses in fruit
industry during the storage process caused by infected fruits.
Better results have been obtained by Li et al.[130] using only four key wavelengths: 575, 698, 810, and
969 nm selected by PCA in order to develop a faster classification method and making it easier to establish
an online MI system. They used a Vis/NIR-HSI system (reflectance mode at 325–1100 nm) to discrimi-
nate between uncontaminated and decayed oranges (affected by P. digitatum), obtaining an overall
classification accuracy for training set and test set of 100% and 98.6%, respectively, with no false negatives.
These results significantly improve those obtained by Folch-Fortuny et al.,[31] providing an addi-
tional alternative for the citrus fruit decay detection problem. Nevertheless, despite these good results,
according to the authors, further research is still needed to establish online MI system for industrial
applications, as well as verify performance of the proposed algorithm to detect early decay caused by
other fungal species.
Furthermore, the most frequent signs of microbial deterioration in fruits are the reduction of sugar
contents, elevated acidity, and superficial growth of aerobic bacteria, yeasts, and molds.[133] To that
respect, Teena et al.[22] evaluated the growth of A. flavus in date fruits and their results revealed that the
symptoms of contamination were visible only from the sixth day of infection. At the same time (for 10
days) they evaluated the date fruits by HSI (960–1700 nm), observing changes in the spectral properties
caused by the chemical modification of constituents, especially a reduction of the sugar content. PCA was
applied to select the most significant wavelengths, and LDA and quadratic discriminant analysis (QDA)
were applied in the statistical classification. Classification accuracies higher than 91% were achieved, and
in most of the cases QDA yielded better accuracy in all the models tested. However, the authors
highlighted that further works are required to test this technique for other fungal species and its effect
on the chemical composition of different fruits.
Previous results suggest that a simple microbial detection system and non-destructive imaging
inspection method may have significant potential to help and ensure food quality and safety in the fruit
and vegetable industries, an aspect of vital importance since in many cases the symptoms of contam-
ination are visible by direct observation just several days after the infection.
Contamination by insects in plant-based foods

Detection of insect damages in plant-based foods is another challenge as they can cause serious
economic loss.[12,134] Non-invasive measurement is a complicated task due to the fact that larvae
generally hide themselves deep inside the fruits and vegetables and they cannot be easily detected
from outside.[9] In Table 3 some studies carried out using HSI technology to detect insect infestation
in different plant based foods are shown.
Xing et al.[135] developed an MI system to evaluate internal insect infestation in cherries by using
Vis/NIR-HSI in reflectance (590–1550 nm) and transmittance (580–980 nm) modes. A GA was
applied to select optimal wavebands in both modes, and the best results were obtained in reflectance
mode in the NIR region. The PLS-DA results indicated that models built with three or four GA
selected wavebands (MI system) gave similar classification accuracy to the HSI model. Nevertheless,
the authors highlighted that due to the stochastic nature of the GA, the efficiency of this MI system
needs to be verified in future works.
Also, Lu and Ariana[58] used HSI in transmittance (740–1000 nm) and reflectance (450–740 nm) modes
to detect fruit fly infestation in pickling cucumbers. PLS-DA was performed to discriminate wholesome
and infested samples, and results showed a better accuracy in transmittance mode (88%–93%). The finding
pointed out that the HSI system offers a better detection than manual inspection (75%), and whose
performance decreased significantly for smaller-size cucumbers.
On the other hand, Singh et al.[136] used NIR-HSI (1000–1600 nm) for detecting insect damage
caused by Sitophilus oryzae, Rhyzopertha dominica, Cryptolestes ferrugineus, and Tribolium castaneum
in wheat grains. The acquired spectral data were reduced by PCA, and by using LDA and QDA, they
correctly classified 85%–100% healthy and insect-damaged wheat grains.
Later, discrimination between insect-damaged (Etiella zinckenella Treitschke) and undamaged
vegetable soybeans was performed by Huang et al.[55] applying hyperspectral transmittance images
technique at wavelengths of 400–1000 nm. Support vector data description (SVDD) algorithm was
used to develop the classification models, achieving a discriminate overall accuracy of 95.6% for the
validation set of samples.
More recently, Mireei et al.[54] have developed a new approach to detect insect infestation in
tomatoes based on an MI system (400–1100 nm). Correlation-based feature selection (CFS) algorithm
was used to find the best wavelengths (733, 746, 821, 911, 944, and 957 nm). To classify tomatoes, three
different machine learning techniques: ANN, SVM, and Bayesian networks (BN) were implemented,
obtaining the best performance by ANN, based on spectral difference features with a classification
accuracy of 95.0%.
Furthermore, insects are an important source of microbial contamination in plants (at larval and adult
stages), for example, the “tomato hornworm,” a common pest affecting tomatoes and other fresh
products, whose excrements can be a vehicle of pathogenic bacteria.[137] Yang et al.[138] used a hyper-
spectral fluorescence line-scan imaging system (400–800 nm) in order to develop a simple MI algorithm
to detect frass contamination at different concentrations on surfaces of mature red tomatoes. Five
wavelengths of 515, 640, 664, 690, and 724 nm were selected to be used in three ratio functions: R515/
640, R724/690, and R664/690, for a multispectral frass-detection algorithm. Results show that this procedure
is capable of detecting over 99% of contaminated samples with 0.2 and 0.1 kg/L frass contamination
spots, which is very difficult to achieve with a simple visual inspection. However, differentiation of 0.05
and 0.02 kg/L frass contamination spots was more difficult. Therefore, the efficiency of this MI system
needs to be improved in future works.
Applications to evaluate biological contaminants in animal-based foods

Microbial contamination in meats and fish
Meats are an excellent source of high-quality nutrients, ensuring a good balance of energy, protein,
vitamins, and minerals. However, they are also an ideal substrate for the growth of both spoilage and
pathogenic microorganisms.[75,139] Spoilage in meats is caused by the growth and enzymatic activity of
microorganisms.[140] As a consequence of deterioration, undesirable metabolites are formed which could
result in off flavours, nutrient loss, changes in appearance, colour, etc. In many cases, consumer-purchasing
decisions depend on meat colour more than any other quality parameters because consumers use
discoloration as an indication of freshness and wholesomeness.[6]
Beyond this, a high microbial population may represent a serious danger to consumer health,
especially in the case of pathogens like Salmonella, Pseudomona, E. coli, Campylobacter, among
others.[74,101,141,142] Furthermore, there is a growing trend to use HSI to measure total microbial
counts, Enterobacteriaceae, Pseudomonas, E. coli, and lactic acid bacteria loads,[12,14] due to the
sufficient spectral information obtained from the samples and their correlation with traditional
microbial count methods.[141,143,144] Enterobacteriaceae is a group of pathogens including E. coli,
Shigella, Salmonella, or Yersinia, whose presence is used as an indicative of hygienic practices.
Table 4 shows some applications of HSI for detecting microbial contamination in different types of
meat and marine products.
Microbial contamination in beef and pork meat

Peng et al.[74,145] evaluated microbial contamination in beef steaks applying HSI (400–1100 nm) and
total viable count (TVC) methods. A multiple linear regression (MLR) model was established to
predict log10 TVC, obtaining an R2p = 0.95. Besides that, they observed a relation between meat
Table 4. Hyperspectral imaging (HSI) for detecting viable microorganisms and parasites in meat and marine products.
Wavelength
Contaminant Food Modea (λ, nm) Accuracy References
Fecal material and ingesta Poultry carcasses R 400–1000 89–98% [158]
Poultry carcasses R 400–900 96.2% [161]
Organic residues on F 500–700 97.5–100% [168]
equipment surfaces in poultry
processing plants
Escherichia coli Pork S 400–1100 R = 0.88 [142]
Pseudomona Chicken meat R 910–1700 R = 0.87 [101]
Enterobacteriaceae Chicken meat R 900–1700 R2 = 0.87 [141]
Salmon R 900–1700 R = 0.95 [181]
LAB Salmon R 900–1700 R = 0.940, 0.925 [172]
EPC Salmon R 900–1700 R = 0.964, 0.953 [180]
TVC Chicken meat R 900–1700 R = 0.94 [143]
Chicken meat R 400–1000 R2 = 0.6833 [163]
Pork R 400–1100 R2 = 0.987, 0.942 [149]
Pork R 400–1100 R2 = 0.9236 [150]
Pork R 900–1700 R2 = 0.82 [152]
Pork R 400–1100 R = 0.886, 0.863 [154]
Beef S 400–1100 R2 = 0.95 [74]
Beef R 405–970 R2 = 0.98 [79]
Beef S 400–1100 R2 = 0.96 [145]
Salmon R 400–1700 R2 = 0.985 [144]
Carp R 400–1000 R2 = 0.90 [178]
TBC Salmon I 400–1000 88% [170]
Parasites Cod I 400–1000 60.3–70.8% [62]
Cod T 400–1000 58% [190]
Clam T 400–1000 85% [192]
LAB, lactic acid bacteria; EPC, Enterobacteriaceae and Pseudomonas count; TVC, total viable count; TBC, total bacterial count; R,
correlation coefficient; R2, coefficient of determination.
a
R, reflectance; S, scattering; T, transmittance; I, interactance; F, fluorescence.
spoilage and oxidation of haemoglobin and myoglobin, which was localized in the absorption bands of
596 and 760 nm, corresponding to the spectral signature of oxymyoglobin and oxyhaemoglobin,
respectively.
Similar findings were reported by Panagou et al.[146] where beef samples were analysed by multispectral
reflectance (405–970 nm) and microbial counts (TVC, Pseudomonas, and Brochothrix thermosphacta)
methods, Pseudomonas being the dominant microbial group due to its fast growth. PLS-DA analysis was
employed for the discrimination in three microbiological quality classes: class 1 (TVC < 5.5 log10 CFU/g),
class 2 (5.5–7.0 log10 CFU/g), and class 3 (TVC > 7.0 log10 CFU/g), obtaining an overall classification rate
for the three quality classes of 91.8% and 80.0% for calibration and validation, respectively. Furthermore,
PLS regression models were developed to provide quantitative estimations of microbial counts during meat
storage, obtaining values of R of 0.90–0.93 and 0.78–0.86 for model development and validation,
respectively.
Additionally, this study revealed that reflectance decreased as the microbial population increased,
especially between 600 and 700 nm, phenomenon related to synthesis of oxymyoglobin, deoxymyoglobin,
and metmyoglobin, in concordance with Peng et al.[74] Also, a decrease in reflectance values at 850 and 970
nm was observed, which could be related with the modification of fat content, whose absorption band is
around 940 nm.[147] However, the authors suggest the need of new models for the determination of specific
microbial groups, as it was performed in other studies,[101,141,142] because the counts of TVC does not fully
reflect the spoilage dynamics in meat.
More recently, Tsakanikas et al.[79] have employed a multispectral system in reflectance mode (405–970
nm) for the discrimination of beef samples in two quality classes: TVC < 2 log10 CFU/g and TVC > 2 log10
CFU/g (mix of mesophilic and psychrophilic bacteria), obtaining classification rates over 80% at different
storage temperatures using Gaussian mixture models (GMM). Moreover, the calculated R2 to predict
counts of TVC from the spectral information was 0.98, as it was estimated using a support vector machine
regression (SVMR) model. The authors addressed the need for validation of developed models in different
storage conditions, since different temperatures favour the growth of specific microorganisms.
Regarding pork, Peng and Wang[148] applied HSI (400–1000 nm) to detect bacterial contamina-
tion, determining five optimal wavelengths by stepwise discrimination method: 480, 525, 650, 720,
and 765 nm. Least square support vector machine (LS-SVM) was adopted as the modelling method
to predict the TVC, and best TVC prediction results were observed taking into account the data for
the five optimal wavelengths with an R = 0.87. The proposed method demonstrated better results
than ANN or MLR methods.
Likewise, Wang et al.[149] by using an LS-SVM method obtained an R2P of 0.9426 in a study performed
to predict the TVC of fresh pork meat based on HSI data. Also based on LS-SVM, Wang et al.[150]
developed a model from eight selected wavelengths (477, 509, 540, 552, 560, 609, 720, and 772 nm),
obtaining a similar result (R2P = 0.9236).
Later, Dissing et al.[151] used a MI system in 18 different wavelengths (405–970 nm) for spoilage
degree detection of pork. In addition, a sensory evaluation panel composed of five members was
recommended to judge the spoilage degree into one of three classes (fresh, semi-fresh, and spoiled).
The classification of images in these three categories, and prediction of TVC, was done using a logistic
regression model. Results indicated that the MI system was capable of differentiating the meat samples
with an overall classification rate of 76.13% according to the defined sensory scale. For the microbial
counts, an overall classification performance of 80.0% was achieved. Besides, the TVC value was also
successfully predicted with an RMSEP of 0.551 and a SEP of 7.47%.
For their part, Barbin et al.[152] evaluated the effectiveness of NIR-HSI (900–1700 nm) in predicting
microbial contamination in pork. They applied PLSR, obtaining values of R2P = 0.82 for TVC, and R2P
= 0.85 for psychrotrophic plate counts (PPC), as well as for the determination of contamination maps
(chemical images) of pork samples stored at different temperatures. Alongside, they were able to
discriminate between both fresh and contaminated meat with 95% and 93% of accuracy, respectively,
using LDA model. They also observed an increase in absorbance values in the contaminated samples
between 1300 and 1600 nm, as result of protein degradation by contaminating microorganisms.
More recently, Ma et al.[153] applied an MI system utilizing 19 different wavelengths (400–1000 nm) to
determine aerobic plate counts (APC) in cooked pork sausages stored at 4°C. PLSR was applied to
establish prediction models for APC, obtaining an R2 of 0.89. The prediction model was then transferred
to each pixel in the image for visualizing the distribution of APC of the samples. According to the
authors, future studies with more samples under different technological or storage conditions and types
of packages should be studied to establish more accurate and robust detection models which can be
applied in the meat industry.
The versatile application of HSI would seem to offer numerous potential advantages, including reduced
labour costs, the elimination of human error and/or subjective judgment, and allowing a real-time product
data creation and document generation reliable for traceability and labelling in the meat industry.[152]
However, in spite of good results, the presence of different micro flora would lead to variations in model
precision,[154] which indicates the need of new models for the determination of specific microbial groups,
because some of these analytical methods, mainly TVC, does not fully reflect the spoilage dynamics.
Therefore, HSI is an interesting technological alternative for identification of specific bacterial species. For
instance, Tao et al.[142] used an HSI system (scattering mode at 400–1100 nm) to determine E. coli
concentration in pork meat by using an MLR model, obtaining a reasonable performance with an RCV
of 0.88.
Microbial contamination in chicken meat

Current contaminated pieces detection procedures are mainly conducted by human visual perceptions,
which is both labor-intensive and prone to both human error and inspector-to-inspector variation.[7,155]
HSI has been used to detect chicken meat contamination with pathogenic microorganisms based on
spectral differences between contaminated meat and meat from healthy chickens.[156,157]
It is known that most research works related to the HSI detection of meat contamination by organic
residuals were carried out on chicken meat, where contamination by pathogenic bacteria can potentially
occur as a result of exposure of the pieces to fecal and/or digested materials during or after slaughter.[6,7]
However, many of these studies have not been evaluated in real-time applications.[158] It is estimated that
only 5 mg of fecal material can pose a danger to humans due to its high pathogenic bacteria load such as
Campylobacter, Shigella, Salmonella, Yersinia, or E. coli,[141,159] so that early detection can prevent
adverse effects on consumer health and quality. HSI is the tool that provided the best results in this
regard.
Windham et al.[160] extracted four optimal wavelengths: 434, 517, 565, and 628 nm using principal
component (PC) loading weights, and developed effective hyperspectral image processing algorithms,
specifically band ratio of 565/517 for the identification of contamination in poultry carcasses, being able
to detect 99% of the fecal contaminants using single-term linear regression (STLR). The same ratio was
used later in a similar study, obtaining a prediction accuracy of 96.4% at wavelengths between 430 and
900 nm.[161]
Likewise, Chao et al.[156] developed a system to classify fecal contaminated from uncontaminated
chicken meat at a high-speed processing (140 pieces per minute), applying HSI at 580 and 620 nm
(since these wavelengths showed the greatest difference between the average wholesome and average
unwholesome chicken spectra). Similar results were obtained in a later study of in-plant real-time
detection, in which they identified fecal spots on the carcasses at 140 pieces per minute, and the
detectable feces could be as little as 10 mg.[162]
A similar study was developed by Yoon et al.[158] in an online real-time HSI system (400–1000 nm).
The fecal detection algorithms using 517, 565, and 802 nm allowed to differentiate fecal and digested
material contamination on fresh cuts of chicken meat at a speed of 140 and 180 pieces per minute, with a
prediction accuracy of 98% and 89%, respectively, with minimum false-positive errors (less than 1% in
some cases). Besides that, they provided a commercially viable imaging platform for fecal detection.
More recently, a new two-band freshness index (TBFI) was used by Ye et al.[163] to develop a
bacterial prediction model (HSI vs. TVC) with an R2P of 0.6833, based on the wavelengths 650 and 700
nm [TBFI = (Rλ1 – Rλ2)/(Rλ1 + Rλ2)]. Additionally, HSI has been also implemented to detect specific
microbial groups such as Enterobacteriaceae and Pseudomonas on chicken fillets, correlating with the
traditional microbiological plate count techniques.
On the other hand, Feng and Sun[143] evaluated the effectiveness of NIR-HSI (910-1700 nm) to predict
contamination in chicken meat. Ten wavelengths were selected for the simplified PLSR model based on
absorbance spectra (AS-PLSR): 961, 1054, 1081, 1084, 1191, 1198, 1201, 1208, 1218, and 1328 nm. The
values of R and RMSEs for this model were 0.96 and 0.40 log10 CFU/g (calibration) as well as 0.94 and
0.50 log10 CFU/g (cross-validation), respectively. Meanwhile the residual predictive deviation (RPD)
value was 2.75 (Table 5). When the best PLSR models (both full wavelength or simplified models) were
finally confirmed, they were applied for predicting bacterial loads in each pixel of the sample images
(chemical image).
Feng et al.[141] applied NIR-HIS (900–1700 nm) for determination of Enterobacteriaceae loads on
chicken fillets. They developed simplified models (MI system) by applying second derivative spectra and
weighted PLS regression coefficients to select three key wavelengths: 930, 1121, and 1345 nm, obtaining
values of R2 of 0.89, 0.86, and 0.87 and RMSEs of 0.33, 0.40, and 0.45 log10 CFU/g for calibration, cross-
validation, and prediction models, respectively. The prediction map (chemical image) provided a good
approach for visualizing the distribution of Enterobacteriaceae. Moreover, an increase in spectral
reflectance as the microbial load increased was observed, due to the changes caused by microbial
metabolism.[164]
Also, Feng and Sun[101] correlated the spectral information obtained by HSI (900–1700 nm) with
traditional counts of Pseudomonas in chicken fillets by using PLSR. To enhance the model, they
selected 14 wavebands in five spectral segments (1138–1155, 1195–1198, 1392–1395, 1452–1455, and
1525–1529 nm) based on genetic algorithm (GA), producing R and RMSEs of 0.91 and 0.55 log10 CFU/
g, 0.87 and 0.65 log10 CFU/g, and 0.88 and 0.64 log10 CFU/g for calibration, cross-validation, and
prediction models, respectively.
Furthermore, the rapid growth of Pseudomonas is responsible for the spoilage, exerted in substrates
such as glucose to synthesize 2-oxo-gluconate and gluconate,[165,166] as well as an increase of ammonia
production and pH, as a result of amino acid metabolism,[167] hence the formation and increased
concentrations of these substances causing undesirable odours and tastes.[166]
According to these studies,[101,141,143] the full-wavelength models (HSI systems) compared with the
simplified models (MI systems) showed better statistical indicators (R/R2 and RMSE, Table 5). Therefore,
already established MI system could be more suitable for online applications in agreement with its higher
robustness, relatively low instrumental cost, and high analytical speed.[4–6,9] Nevertheless, according to
the authors, more studies should be carried out to further verify the effectiveness of HSI and to figure out
the feasibility of MI systems for real-time prediction of Pseudomonas and Enterobacteriaceae loads in
chicken or other meat.
On the other hand, organic residues including fat, blood, and feces on equipment surfaces in poultry
processing plants can generate cross-contamination and increase the risk of unsafe food for consumers.
Qin et al.[168] applied a line-scanning HSI system (500–700 nm), and two soft independent modeling by
class analogy (SIMCA) models were developed to differentiate organic residues and stainless steel
samples: two-class (“stainless steel” and “organic residue”) and four-class (“stainless steel,” “fat,”
“blood,” and “feces”), whose classification accuracies were 100% and 97.5%, respectively, identifying
an optimal single band (666 nm, false-negative errors for chicken blood) and a band-pair ratio (503/666,
for detecting various chicken residues on stainless steel surfaces) by correlation analysis.
Similarly, Jun et al.[169] carried out a study for detecting microbial biofilms of pathogenic E. coli and
Salmonella on food-contact surfaces such as stainless steel, high-density polyethylene (HDPE), plastic
laminate (formica), and two variations of polished granite, by using hyperspectral fluorescence imaging
(421–700 nm). PCA was used to reduce the dimensionality of the data, and the result showed an overall
rate of 95% for detecting biofilm spots on stainless steel, HDPE, and granite. However, too many false
positives were present to accurately determine an effective biofilm detection rate on formica. This could
be due to the lower microbial density observed on formica (approximately 4.3–6.4 log CFU/cm2).
Besides, a single band image at 559 nm was able to detect the biofilm spots on stainless steel surfaces.
Table 5. Multispectral imaging (MI) models developed from hyperspectral imaging (HSI) models by selecting key wavelengths.
HSI (full wavelengths) MI (optimized model)
Wavelength
Food sample: contaminant (λ, nm) Statistical indicators Selected wavelengths Statistical indicators References
Wheat grains: mildew 400–1000 nm Calibration model: R2C = 0.89, Four wavelengths: 450, 561, 861, and 917 nm Calibration model: R2C = 0.89, [32]
damage RMSEC = 0.67 RMSEC = 0.68
2
Validation model: R2V = 0.87, Validation model: R V = 0.87,
RMSEV = 0.73 RMSEV = 0.74
Chicken fillets: Pseudomona 900–1700 nm Calibration model: R = 0.88, Fourteen wavebands in five spectral segments: Calibration model: R = 0.91, [101]
loads RMSE = 0.60 log10 CFU/g 1138–1155, 1195–1198, 1392–1395, 1452–1455, RMSE = 0.55 log10 CFU/g
Cross-validation model: R = 0.83, and 1525–1529 nm Cross–validation model: R = 0.87,
RMSE = 0.71 log10 CFU/g RMSE = 0.65 log10 CFU/g
Prediction model: R = 0.81, RMSE Prediction model: R = 0.88, RMSE
= 0.80 log10 CFU/g = 0.64 log10 CFU/g
Chicken fillets: 930–1450 Calibration model: R2 = 0.88, Three wavelengths: 930, 1121, and 1345 nm Calibration model: R2 = 0.89, [141]
Enterobacteriaceae loads RMSE = 0.35 log10 CFU/g RMSE = 0.33 log10 CFU/g
Cross-validation model: R2 = 0.82, Cross–validation model: R2 =
RMSE = 0.45 log10 CFU/g 0.86, RMSE = 0.40 log10 CFU/g
Prediction model: R2 = 0.85, Prediction model: R2 = 0.87,
RMSE = 0.47 log10 CFU/g RMSE = 0.45 log10 CFU/g
Chicken fillets: TVC 910–1700 nm Calibration model: RC = 0.97, Ten wavelengths: 961, 1054, 1081, 1084, 1191, Calibration model: RC = 0.96, [143]
RMSEC = 0.37 log10 CFU/g 1198, 1201, 1208, 1218, and 1328 nm RMSEC = 0.42 log10 CFU/g
Cross-validation model: RCV = Cross-validation model: RCV =
0.93, RMSECV = 0.57 log10 CFU/g, 0.93, RMSECV = 0.54 log10 CFU/g,
RPD = 2.60 RPD = 2.75
Salmon: LAB 900–1700 nm RP = 0.929, RMSEP = 0.515 log10 Eight wavelengths: 1155, 1255, 1373, 1376, 1436, RP = 0.925, RMSEP = 0.531 log10 [172]
CFU/g 1641, 1665, and 1689 nm CFU/g
Salmon flesh: 900–1700 nm RP = 0.954, RPD = 3.313, RMSEP = Nine wavelengths: 931, 1138, 1175, 1242, 1359, RP = 0.964, RPD = 3.715, RMSEP = [180]
Enterobacteriaceae loads 0.481 log10 CFU/g 1628, 1641, 1652, and 1655 nm 0.429 log10 CFU/g
Salmon flesh: 900–1700 nm RP = 0.94, RPD = 2.97, RMSEP = Eight wavelengths: 924, 931, 964, 1068, 1262, 1373, RP = 0.95, RPD = 3.23, RMSEP = [181]
Enterobacteriaceae loads 0.53 log10 CFU/g 1628, and 1668 nm 0.47 log10 CFU/g
Carp: Escherichia coli loads 400–1000 nm R2P = 0.88, RMSEP = 0.26 log10 Six wavelengths: 424, 451, 545, 567, 585, and 610 R2P = 0.87, RMSEP = 0.27 log10 [182]
CFU/g, RPD = 5.47 nm CFU/g, RPD = 5.22
R, correlation coefficient; R2, coefficient of determination and the corresponding root mean square error (RMSE) in: calibration (RC/R2C, RMSEC), cross-validation (RCV/R2CV, RMSECV), and prediction
INTERNATIONAL JOURNAL OF FOOD PROPERTIES
(RP/R2P, RMSEP); RPD, residual predictive deviation.

S1283
Microbial contamination in fish

Fish and other marine products are highly perishable due to its nutritional richness, which makes them
suitable for the growth of pathogenic and spoilage microorganisms. The growing demand for new
conservation techniques to maintain quality and safety of these products as well as the accomplishment
of vigorous controls at different stages of the distribution chain entails the use of potent analysis
techniques to detect possible contamination.
Deterioration of fish can occur either as the result of autolytic endogenous enzymatic degradation or
by the microbial spoilage,[170] producing undesirable modifications such as textural changes, discolora-
tion, development of off-odours, production of slime, increase of acidity, and formation of toxic
metabolites.[171–174] The presence of aerobic microorganisms can promote unstable oxygen derivatives
that can accelerate lipid oxidation and pigment oxidation such as haemoglobin and myoglobin,[175–177]
and thereby generating changes in the colour and other characteristics. In recent years, HSI has been
extensively studied (Table 4) and implemented as a technological alternative to traditional analytical
methods and has the proven potential to detect the presence of biological contaminants in fish.[5]
Sone et al.[170] used Vis/NIR-HSI (400–1000 nm) and total bacterial counts (TBC) methods to
assess bacterial contamination degree on salmon fillets. K nearest-neighbour (Knn) classifier was
used for the classification of fillets. The best classification was achieved using five single wavelengths:
606, 636, 665, 705, and 764 nm, with an accuracy rate of 88%. In addition, the authors reported an
increment of absorbance at 636 nm, phenomenon related to the oxidation of haemoglobin and
myoglobin to methemoglobin and metmyoglobin, respectively.[146]
In the same way, Wu and Sun[144] used time series-hyperspectral imaging (TS-HSI) between 400 and
1700 nm to predict contamination in salmon fillets. Competitive adaptive reweighted sampling (CARS)
was conducted to identify the most important wavelengths that had the greatest influence on the TVC
prediction, and eight wavelengths: 495, 535, 550, 585, 625, 660, 785, and 915 nm were selected. On the
basis of these wavelengths, a CARS-PLSR model to predict TVC was developed, with an R2 = 0.985 and
RPD = 5.127. Similar work was carried out by Cheng and Sun[178] to predict TVC in carp fillets by using
HSI (400–1000 nm), observing an increase in reflectance values. Seven optimal wavelengths: 410, 488,
553, 665, 750, 825, and 960 nm were selected by successive projections algorithm (SPA), and a simplified
SPA-PLSR model gave values of R2 = 0.90, RMSE = 0.57 log10CFU/g, and RPD = 3.13.
In both studies,[144,178] the best model was used to predict the TVC values of each pixel within the
region of interest (ROI) of fish pieces (chemical image), and the multispectral (MI) systems were also
recommended to be developed for online applications.
Regarding specific microbial species, among the most important fish-contaminating bacteria are
some species of Enterobacteriaceae or Pseudomonas and lactic acid bacteria (LAB).[179–181] He and
Sun[181] applied HSI (900–1700 nm) for evaluating Enterobacteriaceae contamination of salmon flesh
by correlating the spectral information with plate counts (CFU/g). By applying SPA, eight key
wavelengths: 924, 931, 964, 1068, 1262, 1373, 1628, and 1668 nm were selected to develop a simplified
SPA-PLS model, obtaining values of RP, RMSEP, and RPD of 0.95, 0.47, and 3.23, respectively. The
same research team[180] applied HSI to predict Enterobacteriae and Pseudomonas counts (EPC) in
salmon fillets. The better performance was found with a simplified PLS model, established with nine
key wavelengths: 931, 1138, 1175, 1242, 1359, 1628, 1641, 1652, and 1655 nm, obtaining values of R,
RMSE, and RPD of 0.964, 0.429, and 3.715, respectively.
Similar results were obtained in their previous study[172] aimed to predict LAB loads in salmon fillets
by NIR-HIS. Eight important wavelengths: 1155, 1255, 1373, 1376, 1436, 1641, 1665, and 1689 nm were
selected using a CARS algorithm, and an optimized CARS-LS-SVM model was established, leading to RP
of 0.925 with RMSEP of 0.531 log10 CFU/g. Besides, the authors concluded that an increase in spectral
reflectance is directly proportional to the bacterial counts. Also, Cheng and Sun[182] evaluated E. coli
contamination in carp flesh using HSI (400–1000 nm), and values of R2P and RDP over 0.87 and 5.22,
respectively, were obtained by application of PLSR and MLR models by mining either full-wavelength
spectra or six selected key wavelengths: 424, 451, 545, 567, 585, and 610 nm.
In all cases, the best model was transferred to each pixel of images, and colourful distribution maps
(chemical image) with different colours and representing different numbers of Enterobacteriaceae, EPC,
or LAB counts were produced. Given the promising results, the simplified models (MI systems) showed
better statistical indicators (R/R2, RMSE, and RPD, Table 5). Therefore, the developed MI system could
be more suitable for online applications, due to its higher robustness, relatively low instrument cost, and
high analytical speed.[4–6,9] However, more studies are still required to refine its applicability at an
industrial level.
Microbial contamination in eggs

The most studies aimed to evaluate this type of food by using HSI have been applied to inspect
quality attributes such as polyunsaturated fatty acids content (omega-3),[183] freshness, bubble
formation, or scattered yolk.[184] There is no comprehensive information available regarding the
application of HSI to evaluate microbial contamination in these food products.
It is known that organic residues on eggshells such as blood, feathers, feces, etc., can be a vehicle for
transmitting pathogens such as Salmonella, Enterobacter, or Staphylococcus,[185] hence their early
detection is the most important way to identify contaminated products and prevent future risk. To
that respect, Lunadei et al.[186] used MI (440–940 nm) to discriminate clean and contaminated eggs
with organic residues. They employed an image algorithm based on a combination of red (700 nm) and
blue (450 nm) images, achieving a classification rate near 98% based on the geometric characteristics of
the detected stains, with a very short processing time (0.05 s). Afterwards, the system was validated
using a second set of samples, obtaining a classification rate of 97%.
Given a simple algorithm based on only two wavelengths, the proposed method is a cheap, easy,
accurate, and fast alternative that could be implemented in an online process. However, in spite of
the obtained results, further research is necessary in order to optimize the accuracy, for example,
employing samples with different kinds of defects and monitoring whether changes in the illumina-
tion system affect the results of the imaging process.
Evaluation of parasitic contamination

Table 4 shows some of the studies carried out by HSI application for detecting parasites in different types
of meat and marine products. The presence of parasites, such as Anisakis simplex in fish or Trichinella
spp. and Taenia solium in pork and beef, is related to the incidence of many diseases.[187–189] Parasitic
infection is a severe food safety challenge, and it is important for the food industry to rely on compatible
preventive action and online detecting methods to avoid the presence of parasites.[64] For example, fish
fillets are inspected by transillumination on candling tables and parasites are removed manually by
trained personnel,[60] achieving to identify up to 70% of contaminated pieces.[61] However, this proce-
dure and other options have disadvantages as previously mentioned.
Another alternative to assess the presence of parasites in fish is the application of image analysis
techniques. Although it is sometimes invisible to human eyes, parasites could be easily detected by HSI
due to the fact that their presence in fish flesh present distinctive “spectral fingerprints” compared with
normal fish muscles.[6] In this regard, a pioneering study was developed by Wold et al.,[77] who
investigated how MI (transmittance mode at 400–1000 nm) in combination with SIMCA classification
can be used for automatic detection of parasites in cod fillets, detecting nematodes (Gadus morhua) to
a depth of 6 mm. However, despite promising results, the authors recommended further studies to
verify feasibility for the fish industry.
Heia et al.[64] achieved a greater depth detection of nematode G. morhua in cod fillets using HSI
(350–950 nm). The spectral images were analysed by discriminant partial least square (DPLS)
regression in order to identify pixels representing fish sample with embedded nematodes. This
method demonstrated the great potential of HSI to detect parasites to a greater depth (8 mm), which
is 2–3 mm deeper compared with the classic visual inspection,[60,61] besides the method is non-
intrusive and should thus be feasible for industrial purposes.
For their part, Sivertsen et al.[190] applied HSI (400–1000 nm) to evaluate the presence of parasites in
cod fillets using a mobile system at an operating speed of 25 mm/s, obtaining in some cases better
results than the visual inspection. The Gaussian maximum likelihood (GML) classifier achieved a
detection rate of 50.7% for Anisakis simplex and 65.3% for Pseudoterranova decipiens. However, the
system could not process the fillets with skin and the speed was slow. Later on, the same authors[62]
optimized the application of HSI in order to detect the presence of the same nematodes at a required
industrial speed of 400 mm/s in cod fillets, and the GML classifier improved the detection rates to
60.3% (A. simplex) and to 70.8% (P. decipiens). In addition, these parasites were classified based on the
depth or the section of fish piece where they are localized. However, the authors suggest the need to
improve the depth of detection of parasites and the correct detection rate in future applications.
It is also reported the possible use of HSI to evaluate the presence of non-pathogenic parasites
such as Edotea magellanica, which is not risky to human health but its presence affects food quality
standards and generates rejection from consumers.[191] In this regard, Coelho et al.[192] working with
a HSI system (400–1000 nm), and a summary of the spectral information contained at the
wavelengths 624.1, 760.4, and 888.6 nm, achieved 85% of detection accuracy to predict the presence
of this parasite in cooked clam. Besides, they observed a decrease in transmittance between 600 and
950 nm, which was attributed to the spectral properties of the parasite, especially at 720 nm.
As seen so far, the different research works regarding HSI application for detecting the presence of
parasites are related to the assessment of fish and other marine products, while there is not comprehensive
information available in the bibliography regarding the application of HSI in other type of meats. One
similar work in this regard was performed by Gómez-De-Anda et al.,[193] using spectroscopy to evaluate
Trichinella spiralis in pork. They were able to discriminate between both infected and non-infected meat by
using SIMCA. In all cases, the recognition and rejection rates were 100% and a higher concentration of
trichinosis in the masseter muscle was detected, suggesting that it could be considered as the best muscle for
parasite identification purposes in contaminated pork.
Finally, it should be noted that there is no information available in the bibliography regarding the
application of HSI to detect Taenia solium (tapeworm), one of the parasites most prevalent in Latin
America, whose infections can lead to “cysticercosis” disease as a result of raw or undercooked pork
consumption.[187,194]
Current and future challenges

Advantages and disadvantages of hyperspectral imaging
This review gives special prominence to HSI as a promising non-destructive technology for the evaluation
of contamination of biological origin in foods, as it offers several improvements, such as speed, accuracy,
and reliability over other methods. Besides, no sample preparation is required; it is a chemical-free
analytical method that does not bring environmental pollution,[5] and is capable to determinate several
features simultaneously in the same sample.[7,15,53] In addition, this non-destructive and non-invasive
technology has potential to realize quantitative and qualitative analysis as well as rapid real-time and online
detection,[11] aspects that with other technologies are not always possible.
However, HSI technology still has some disadvantages that need to be resolved for further applica-
tion at the industrial level. For instance, HSI systems need accurate reference calibration and robust
model transfer algorithms, and do not have good detection limits compared with chemical-based
analytical methods.[11] Moreover, hyperspectral images contain much unnecessary information than a
single-color image and therefore require more processing time to obtain valuable information. Besides
that, the hardware speed of an HSI system needs to be enhanced to satisfy the fast acquisition and
analysis of the enormous hyperspectral data. So, the HSI technology would not be yet advisable for
direct achievement of the online purpose.[5]
To overcome these disadvantages, current studies are aiming to develop models that: (a) allow a rapid
data discrimination and retrieve interesting information, given the large amount of data from the wide
range of spectral bands in which the HSI works, and (b) identify optimal wavelengths for each food or
food constituents, affected by biological contaminants. This is possible due to the potential of HSI to
identify and discriminate different constituents,[76] biological contaminants,[110] or metabolites produced
by contaminant microorganisms, such as toxins[42,45,100,106] or other substances causing undesirable
attributes in food products.[109,172–174]
Future challenges
On the basis of aforementioned disadvantages, scientific and technological challenges that must be
overcome for further application of HSI at the industrial level are
(a) Further research for detecting biological contaminants in liquid foods is needed,[15] since
most applications have been focused on solid samples (Tables 2–4).
(b) Quantification of low concentrations of microorganisms, since most studies have been
focused on assessing microbial densities higher than 102 CFU/g.[2]
(c) Current studies will need to be extended to other microbial species,[2,92] since most of these
studies are limited in detecting and quantifying metabolites produced by microbial
contaminants.
(d) Advanced methodologies that allow discrimination between biological contaminants based on
its specific “spectral signature.”
(e) Optimize the existing models or create more robust models that allow to predict biological
contamination from the spectral data obtained, without running the risk of losing valuable
information.
(f) Validation of developed models on several storage conditions, simulating real life, for
example, at different storage temperatures.[79]
(g) Design of techniques able to fuse the spatial and spectral data, since most of available
hyperspectral data processing techniques focus on analysing the spectral data without incor-
porating information on the spatial data.[3]
Besides, the implementation of online applications, due to the big size of hyperspectral images,
requires efficient technologic tools to analyse such images in a real-time mode. The most hyperspectral
raw data are currently used in off-line systems at laboratory level to select key wavelengths for building
MI systems suitable for online applications.[6,10,14] Therefore, it is needed to develop a fast and efficient
technological support for processing the spectral information, which is considerably disadvantageous
due to the high cost to introduce HSI at an industrial level. An interesting alternative could be the
integration of image-processing algorithms into specialized hardware, then reducing significantly the
test time, as well as development of MI systems for industrial applications due to its relatively low
instrument cost and high analytical speed.[8,9]
Compared with the full-wavelength models (HSI systems), the simplified models (MI systems)
turned out to be more robust. Some works carried out in this respect are shown in Table 5, in which
the improvement of models based on MI can probably be attributed to the elimination of the
uninformative or even misleading wavelengths, and small differences in R/R2, RMSE, and RPD for
models were observed. In addition, the reduction of spectral multicollinearity could also be part of
the reasons for model enhancement.[14]
Likewise, some disadvantages in analysis of microorganisms concerning food quality could be
overcome with the technical integration of HSI with fluorescence microscopic imaging (FMI) and
Raman microscopic imaging (RMI), improving the capability for inspection of bacterial contamina-
tion. In this sense, Chen et al.[4] anticipate that future technological development in the HSI system
for food-safety analysis will promote some novel imaging techniques, such as hyperspectral FMI or
hyperspectral RMI.
On the other hand, HSI systems cannot acquire the deeper constituent spectral information inside
certain samples; penetration depth of the incident radiation will generally be a function of employed
wavelength,[15] for example, in meat samples. This disadvantage could be overcome by identifying
specific spectral bands. Heia et al.[64] detected parasites at a depth of 8 mm at wavelengths between
350 and 950 nm, using the spectral difference between the parasite and the contaminated food. It could
also overcome the disadvantages of similar technologies, such as infrared spectroscopy Fourier transform
with attenuated total reflectance (ATR-FTIR), whose penetration depth of incident radiation is approxi-
mately 1 μm, limiting its application to surface analysis.[195] Additionally, according to previous results
obtained by Sivertsen et al.,[62,190] transmittance mode allowed to obtain better results in detecting
parasites, as well as the interactance mode, which has less surface effects and reduces the influence of
thickness.[12]
Moreover, in the case of plant-based foods, most of the research is being conducted to evaluate specific
cultivars. Obviously such results are variety dependent and cannot be used to evaluate different varieties. So,
future studies should focus on the use of HSI technology for detecting microbial contamination in different
varieties.[92] From a practical point of view, this would imply to build different systems incorporating
different wavelengths depending on the variety.[31] Finally, regarding high cost of the HSI systems
compared with other technologies, this is expected to be a minor barrier in the next years because the
increase of commercial suppliers could reduce the cost and improve its availability.[6] In the last years,
commercial HSI/MI systems for food applications such as Hyperspec Inspector (Headwall Photonics,
Fitchburg, USA) or SisuCHEMA (Specim, Oulu, Finland) are appearing in the market,[10] and recently,
Goel et al.[196] have developed a prototype capable of capturing hyperspectral images with a low-cost
camera (called HyperCam), which uses 17 wavelengths that are spread between 450 and 990 nm, with a cost
of about US$ 800, but could reach US$ 50 to fit on mobile phones.
Critical appreciation and conclusions

Of the numerous applications of HSI, there are different studies aiming to determine their suitability
for detecting contaminants of biological origin, which can cause deterioration of the product at
different stages in the food production chain that will pose a danger to quality products and/or to
consumer health. Contamination is very important at any level, which includes the production of
agricultural raw materials of both animal and plant origin, the industrial processing, packaging and
the storage conditions.
A number of review papers about application of HSI published in the last years are primarily
associated with quality evaluation of food products, however, our work is focused to review the most
notable application of HSI for detecting: (a) viable microorganisms that cause damage to different
levels, such as fungi, bacteria, or viruses that can attack agricultural crops and cause losses in
production and quality, and once harvested may retain residual contaminants that pose a further
risk; (b) microorganisms causing deterioration and quality loss in ready-to-eat foods, reaching certain
levels of microbial population, specially pathogens and/or their toxins, which can be a danger to the
health of consumers; (c) different organic residues such as feces, digestive material, etc., which can be a
vehicle for different microbial contaminants; and (d) parasites, applied mainly to marine products. All
of them, individually or in combination, pose a danger to the quality of the product and/or a risk to
consumer health and safety.
On the other hand, some disadvantages are also presented and discussed. Further research on the
application of HSI to a larger number of microbial species, liquid foods, presence of parasites in other
types of meat (such as pork), and development of models on several storage conditions is needed, which
together with the technical integration of HSI with other technologies such as FMI and RMI, the increase
of commercial suppliers that can reduce the cost and improve the availability of HSI systems, techno-
logical improvements for processing spectral information and search for robust and optimized models,
without running the risk of losing valuable information, would give this technology a better chance of
industrial application as an alternative to traditional techniques such as the liquid chromatography, the
MID-FTIR spectroscopy, or the ELISA and PCR tests, which besides being tedious, are expensive and
their application is limited on the laboratory level. Finally, although many researches for evaluating
biological contamination in foods have been performed, much studies are still needed to standardize the
procedure of HSI in terms of data mining, MI development, and further real-time and online applica-
tions along the various stages in the food production chain.
Funding
This study was funded by the following institutions: Programa Nacional de Innovación para la Competitividad y
Productividad Innóvate Perú – Ex-FINCyT (Contract 407-PNICP-PIAP-2014) and Universidad Nacional de
Trujillo – UNT (PIC2-2013/UNT).
ORCID
Raúl Siche http://orcid.org/0000-0003-3500-4928
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Evaluation of Biological Contaminants in Foods by Hyperspectral Imaging A Review

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International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: https://www.tandfonline.com/loi/ljfp20

Evaluation of biological contaminants in foods by

Ricardo Vejarano, Raúl Siche & Wendu Tesfaye

To link to this article: https://doi.org/10.1080/10942912.2017.1338729

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Evaluation of biological contaminants in foods by hyperspectral

ABSTRACT ARTICLE HISTORY

Figure 1. Main parameters controlled in food production.

Traditional techniques used to detect biological contaminants in foods

Evaluation of viable microorganisms

Evaluation of microbial toxins

Techniques for detection of insect infestation

Evaluation of contamination by parasites

Hyperspectral imaging technology

information of such constituents in different spectral bands, from ultraviolet–visible (UV–vis) to

Applications to evaluate biological contaminants in plant-based foods

Contamination by microbial toxins in plant-based foods

Contamination by viable microorganisms in plant-based foods

Viable microorganisms in cereals

Viable microorganisms in fruits and vegetables

Contamination by insects in plant-based foods

Applications to evaluate biological contaminants in animal-based foods

Microbial contamination in beef and pork meat

Microbial contamination in chicken meat

(RP/R2P, RMSEP); RPD, residual predictive deviation.

Microbial contamination in fish

Microbial contamination in eggs

Evaluation of parasitic contamination

Current and future challenges

Critical appreciation and conclusions

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