International Journal of Food Properties

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

Evaluation of biological contaminants in foods by

hyperspectral imaging (HSI): 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 (HSI): A review, International Journal of
Food Properties, DOI: 10.1080/10942912.2017.1338729

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

Accepted author version posted online: 08

Jun 2017.

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 International Journal of Food Properties

Evaluation of biological contaminants in foods by

hyperspectral imaging (HSI): A review

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Ricardo Vejarano1,2, Raúl Siche1,*, Wendu Tesfaye3

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1
Facultad de Ciencias Agropecuarias, Universidad Nacional de Trujillo (UNT). Av. Juan Pablo

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II S/N, Ciudad Universitaria, Trujillo, Peru

2
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Facultad de Ingeniería, Universidad Privada del Norte (UPN). Av. Del Ejército 920, Trujillo,
Peru
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3
Departamento de Química y Tecnología de Alimentos, Universidad Politécnica de Madrid
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(UPM). Av. Complutense S/N, Madrid, Spain

* Corresponding author: rsiche@unitru.edu.pe (Dr. Raúl Siche); Phone: +51(44)294778.

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Received: 2017-01-16
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Accepted: 2017-06-01
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ABSTRACT

Responding to the ever growing concern about safe foods and security, the food industries are

forced to seek an emerging technology capable of detecting and quantifying contaminations,

especially those of biological origin. Amongst the different emerging technologies,

hyperspectral imaging (HSI) is considered a good alternative as it can be easly applied at all

steps of the food production process and is a non-destructive technique. This paper reviews
targeted analytical applications of HSI in monitoring biological contaminants in food. First,

traditional techniques for detection of biological contaminants in foods are presented, where

disadvantages for practical applications are highlighted and explained in detail in their

respective sections. Second, prominent applications of HSI from the last decade to food safety

and quality assessment are reviewed, especifically focusing on both deteriorative and

pathogenic microorganisms, microbial toxins and parasites; whether acting individually or

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collectively spoil food products and/or represent a health risk to the consumers. Finally, relevant

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current and future challenges, advantages and disadvantages of HSI applications are briefly

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examined.

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Keywords: Hyperspectral imaging, microbial contaminants, toxins detection, parasites

detection, image technology. an

INTRODUCTION
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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
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today’s markets, there is an increasing demand for safe and quality products and also food

safety legislations become more and more stringent.[2]

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The food industry is actually focused on developing innocuous products and requires a constant
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commitment to the design and implementation of procedures and systems to control various

parameters in food products (Fig. 1). Currently available analytical techniques are mostly slow
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methods and destructive. 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.

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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 in determination of chemical and microbiological contamination on food

products.[1,2,14,15]. However, there is no any reference which specifically address 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

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output and quality; microorganisms causing deterioration and quality loss in ready-to-eat foods,

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especially pathogens and/or their toxins; different organic residues that can be a vehicle for

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different microbial contaminants; and parasites.

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It is estimated that between 10% to 50% of agricultural production, especially grains and

vegetables, are lost each year as a result of microbial contamination.[16] Besides, it is estimated
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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
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in 6 Americans (about 48 million people) gets sick, where around 128000 were hospitalized,

and 3000 died of foodborne diseases.[18] Therefore, this review focuses on the most notable
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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,
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packaging and the storage conditions, that is, from-farm-to-table.

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TRADITIONAL TECHNIQUES USED TO DETECT

BIOLOGICAL CONTAMINANTS IN FOODS

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Table 1 summarizes some of the most common techniques for detecting the presence of

contaminants such as bacteria and/or their toxins, fungi and/or their toxins, viruses and parasites

in different foodproducts.

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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 to 3 days for initial results, and up to 7 to 10 days for confirmation.[19] On the

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other hand, early detection of pathogens can be useful to prevent potential negative effects on

consumers’ health and degradation of the food products, that is not always possible by

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conventional plate count, especially with pathogens such as Escherichia coli.[20]

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For its part, the conventional direct microscopic techniques are time-consuming, particularly
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when a large number of samples are analyzed or they do not provide complete quality and safety

assurance responses,[21,22] and do not adequately differentiate between viable and non-viable
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cells. As alternative, methods based on cell staining have been designed,[23,24] which in turn can

be tedious and expensive.

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Meanwhile, the immunology-based methods such as enzyme-linked immunosorbent assay

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(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
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affinity of the antibody to the microorganism or other analyzed 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 limited application at laboratory level[26] and possible

interference of inhibitors present in foods,[27] which could produce erroneous results.

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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 a main viral RNA

encoding G protein in viral hemorrhagic septicemia infection (VHS),[29] one of the most serious

viral diseases damaging both fresh and marine fish species. According to the obtained results,

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these detection techniques are rapid, specific and sensitive. However, most of them are

technically complicated and costly, and require well-trained specialists.[20]

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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
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microbial contaminants.[30] However, among its limitations the difficulty to apply for a wide

variety of microorganisms with different replication time is highlighted for its contribution. This
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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
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bone, glass or metal fragments.[4]

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Regarding to 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
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speeds.[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 to differentiate samples with similar color,

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

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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) spectroscopy[34,35] are not

independent 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,

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information about “where” and “how” microorganisms physically distributed in food samples

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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

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spectroscopy is applicable to characterize homogenous samples by providing mean values of

analyzed samples, so a single measurement may be misleading to represent the bulk value of the
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entire sample when heterogeneous samples are evaluated.[7]
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As a solution to this situation, hyperspectral imaging (HSI) combines the complementary

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approaches of spectral analysis and image processing, thus offering the advantage to evaluate

simultaneously the composition (spectral information) and the external characteristics

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(traditional image analysis) in different foodstuffs.[3,6]

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Evaluation of microbial toxins

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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, very low limit of detection; most of them are generally

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expensive, labor intensive, destructive, time consuming to achieve appropriate results,[45] some

of them are unable to handle a large number of samples,[11] unfriendly organic reagents are

always needed,[46] and their application is also limited at a laboratory level. In addition, the risk

of discarding an entire lot or accepting a contaminated lot as safe due to the unevenly

distribution of contaminated products in storage[47] with the economic losses that it implies.

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Detection of microbial toxins by fluorescence (induced reaction between peroxidase enzyme

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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] although only for

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some 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.
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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
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therefore discarding an entire lot or accepting a contaminated lot as safe.
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Likewise, transient infrared spectroscopy (TIRS) is a technique for on-line, non-contact

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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
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grains. However, the limitation of TIRS is the special requirements on heating the media surface

for producing proper thermal emission,[53] inviable in large batches of food products.
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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 to studies performed on the possible methods for identifying the

insect infestation in agricultural products,[54] although several technologies such as acoustic

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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.[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 discriminate

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between normal and infested tissues.[58] On the other hand, since the insect larvae usually

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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

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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
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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
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infestation in agricultural products.[57,59]
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Evaluation of contamination by parasites

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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]

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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
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performed at room temperature, 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

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use of ultrasonic waves, on the basis to 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 measurement medium, is therefore one of the reason why it has not been

industrialized.

Also, PCR and ELISA has been successfully applied for the detection of parasites in meat,[66-71]

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although with the same disadvantages previously mentioned. For its part, liquid

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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

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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
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unfriendly chemicals,[45,46] and their application is also limited to a laboratory level.
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Because of these disadvantages, the traditional analytical techniques are normally not suitable
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for on-line 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
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as one of the most promising alternatives, which overcomes all of the above-mentioned

disadvantages of conventional technologies used to evaluate foodstuffs, because these methods

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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]
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9
HYPERSPECTRAL IMAGING (HSI) TECHNOLOGY

Also known as “imaging spectroscopy” or “imaging spectrometry”, hyperspectral imaging

(HSI) is an emergent technology that integrates the advantages of spectroscopy and imaging,

allowing to evaluate simultaneously the composition (spectroscopic component) and external

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characteristics (traditional image analysis) of a sample.[3,6,8,11] That is, the spectroscopy respond

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to the questions “what” and “how much” and the image analysis provides an answer to the

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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

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characterisation of a sample.[73] an
Fig. 2a shows a typical laboratory experimental setup of the HSI system. The hyperspectral
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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
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used to characterize the composition of that specific position, and surface-feature information

can be obtained according to the spatial images.[4,11]

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The basic principle of HSI is based on the fact that all materials reflect, scatter or absorb energy
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differently when they are subjected to an electromagnetic radiation source at different

wavelengths 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]

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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).

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Although the constituents of a sample present certain reflectance (or absorbance) values at

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specific wavelengths, as in the case of conventional spectrophotometers, is possible to obtain

complete information of such constituents in different spectral bands, from ultraviolet-visible

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(UV-Vis) to near infrared (NIR),[45] which allows to measure a wide range quality parameters,

as well as the presence of contaminants and their spatial distribution in a given food sample.[77]
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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
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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

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simplicity in sample preparation, and being a chemical-free analysis technology,[15] aspects that

with other technologies is not always possible.

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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. Amongst an increasing number of food

contaminants, those of biological origin may cause a marked accelerated chemical and structural

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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 summarize the outstanding results in the detection and evaluation of

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contaminants of biological origin such as pathogenic microorganisms, undesirable and/or

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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;

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which acting individually or collectively can deteriorate food products and/or represent a health

risk to consumers.
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APPLICATIONS TO EVALUATE BIOLOGICAL

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CONTAMINANTS IN PLANT-BASED FOODS

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Microbial contamination in agricultural crops

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Early detection of crop diseases benefit farmers as they can remove infected plants quickly

before diseases spreads and inflicts further damage.[80] In this sense, adequate monitoring of
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cultivars can be a useful and cost-effective approach to mapping infected crops, for instance, by

the using of hyperspectral airborne imaging. In Table 2 a summary of studies related to spectral

imaging for detecting microbial contamination in different cultivars are presented.

A pioneering study was developed 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 in to account the injuries and color changes in

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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

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the fungal disease known as “tan spot” in wheat, caused by the pathogenic fungi Drechslera

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tritici-repenti. However, according to the authors, the possibilities for fungal disease control

using HSI have to be further investigated.

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On the other hand, contamination by Fusarium ssp. is a serious problem for the cereal industry

due to the potencial capacity for producing mycotoxins. Detection of contamination at the early
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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,
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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
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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
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visible spectrum.
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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

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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%, that could be improved with this index.

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Regarding to fruit crops, Qin et al.[85] applied HSI (reflectance mode at 450-930 nm) for early

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detection of “citrus canker”, a bacterial disease caused by Xanthomonas axonopodis, where

symptoms are visible just after 7 to 10 days after infection. Correlation Analysis (CA) and PCA

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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
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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)
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system for early and real-time “citrus canker” detection.
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The same two-band ratio (R834/R729) selected by CA gave a correlation value of 0.811 and
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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 one-line
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commercial fruit sorting machine that achieved an overall classification accuracy of 95.3%,

operating at speed of 5 fruits/second.

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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] 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

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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

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Discriminant Analysis (SDA) to select key wavelengths that discriminate infected from healthy

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vine leaves in the spectral range between 350-2500 nm, obtaining an overall accuracies of 0.81.

More recently, MacDonald et al.[92] have showed that remote monitoring (hyperspectral airborne

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imaging at 400-1000 nm) of GLRaV-3 infected Cabernet Sauvignon vineyards can be a useful

and cost-effective approach to mapping infected crops, with a success rate of leafroll detection
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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
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other grape varieties, as well as other grapevine pathogens.
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Similarly, early detection of “anthracnose” would avoid a decrease in the strawberry yield
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because not only the healthy and symptomatic stages can be recognized, but also at the

incubation stage before any “anthracnose” symptoms becomes visible. A pioneering study has
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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
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Colletotrichum gloeosporioides to simulate foliar “anthracnose”. Different statistical analysis

methods were evaluated, with the results indicating SDA as the most appropriate method, with

an average accuracy between 80% and 93%.

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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 a 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

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that, the authors propose phenolic compounds as a prominent key discriminating factor between

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virus-infected and healthy seeds where the most common feature of major peaks were consistent

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with the absorption peaks of these compounds, which plants produce to defend themselves not

only against herbivores, but also against competing plants and microorganisms.

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These aforementioned results clearly demonstrate that the use of spectral methods provide
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useful informationns to avoid significant agricultural productivity losses, because it would be

possible to detect infected cultivars before the damage becomes visible.[26,80] Additionally,
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compared to traditional detection methods, HSI offers a potentially valuable alternative for

monitoring crop diseases, since this technology is cost effective, reliable and automatable.[92]
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Contamination by microbial toxins in plant-based foods

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Microbial toxins are among the most controlled food contaminants and its consumption can lead

to health problems such as jaundice, liver carcinomas, oesophageal cancer, neural tube defects,
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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 Aspergillus flavus and Aspergillus parasiticus,[97] ochratoxin produced by Aspergillus,[96,98]

fumonisins and trichothecenes produced by Fusarium,[96] and patulin produced by Penicillium,

Aspergillus or Byssochlamys.[99]

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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 above-mentioned. 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

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detection of the presence of viable microorganisms, and either toxigenic or non-toxigenic strains

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from both bacteria[20,101] and fungi.[22,43] Table 3 summarizes some of the research works carried

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out using HSI technology to evaluate contamination caused by viable microorganisms and/or

their toxins.

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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
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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
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high (>100 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
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aflatoxin concentration in maize grains inoculated with Aspergillus flavus spores, obtaining a R2

of 0.72 using a Multiple Linear Regression (MLR) model. The multivariate analysis of variance
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(α=0.01) showed significant differences between four aflatoxin groups tested: <1, 1-20, 20-100,
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and ≥100 ng/g (parts per billion, ppb), and the classification accuracy ranged 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 Aspergillus flavus (one toxigenic

strain and other non-toxigenic strain) and production of aflatoxin in both germ and endosperm

fractions of grains were evaluated by an affinity chromatography commercial procedure and by

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fluorescence HSI (400-700 nm), thereafter figured out the range between 450 and 500 nm 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

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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

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artificially inoculated maize ears, and two wavelengths (1729 and 2344 nm) were identified to

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characterize aflatoxin exclusively, achieving a higher detection accuracy (92.3%).
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Moreover, applying Vis/NIR-HSI (600-1000 nm) and HPLC analysis, Wang et al.[106] analyzed
M
maize grains contaminated with aflatoxin, and an overall classification accuracy of 98% was

achieved. A combination of PCA and Factorial Discriminant Analysis (PCA-FDA) was

ed

performed to detect and differentiate different levels of aflatoxin, pointed out besides the

detection of this toxin at 735.2 nm as it was obtained by Pearson et al.[102] Similar results were
pt

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
ce

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%.

Ac

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 Aspergillus parasiticus. A two-dimensional Local Discriminant Bases

(LDB) algorithm was developed and a classification accuracy of 92.3% was achieved for

18
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

t
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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

cr
model fitting). Furthermore, on the basis of the predictions provided by a neural network (NN),

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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.
an
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
M

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
ed

1310, 1360, and 1480 nm (to differentiate ochratoxin A contaminated grains). Selected

wavelengths were used as input for statistical classifiers, which differentiated fungal infected
pt

grains with more than 80% (initial periods of infection) and 100% classification accuracy (four
ce

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
Ac

reduced to less than 1 min when it is performed later with selected key wavelengths.

19
Contamination by viable microorganisms in plant-based foods

Safety problems as a consecuence of contaminats of biological origin of most plant-based foods

might be severe due to that a small portion of infected products may contaminate the whole

batches, and ingestion of contaminated products might incur health problems to consumers.[9]

t
Therefore, it is crucial and necessary to apply rapid, accurate, and non-invasive technique in

ip
food analysis procedures to overcome previously mentioned drawbacks. In Table 3 are shown a

cr
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.

us
an
Viable microorganisms in cereals

One of the biggest problems for the cereal industry is the significant loss caused by so-called
M
"storage fungi". If it is not eliminated by proper drying processes after harvest, it may flourish

during storage under favorable conditions,[107,108] affecting important features such as

ed

appearance, chemical composition or germination capacity. Apart from that, this fungal growth

promotes some undesirable side effects as production of toxins and off-odors originated from
pt

the synthesis of volatile compounds such as 3-methyl-1-butanol, 1-octanol and 3-octanone.[109]

ce

The possibility of spectral information for detecting characteristic compounds at certain

Ac

wavelengths is reported by Fernández-Ibáñez et al.,[41] who related the spectral range of 870-

1200 nm with compounds that include the functional group -NH, likely amino acids and

aromatic rings of furans and phenols, formed as a consequence of fungal degradation of cereals.

20
Hyperspectral imaging (HSI) is one of the best alternatives for the identification of different

fungal species, because it shows a specific "spectral signature" for each specie.[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 with 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.

t
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Shahin and Symons,[32] designed a low cost multispectral imaging (MI) system from a HSI

cr
system (400-1000 nm), and reported that 917 nm is a relevant wavelength to detect mildew

us
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
an
(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
M
systems) presents small differences in R2 and RMSE values, and the proposed MI system could

be more suitable for on-line applications, due to its relatively low instrument cost and high
ed

analytical speed.[4-6,9]
pt

Meanwhile, Singh et al.[111] used SW-NIR-HSI (700-1100 nm) for fungal detection in wheat
ce

grains infected by Penicillium spp., Aspergillus glaucus, and Aspergillus niger. Linear

Discriminant Analysis (LDA) classifier was applied and classification accuracy between 97.3-
Ac

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 between toxigenic

and non-toxigenic of A. flavus strains. The prominent dimensionality reduction technique PCA

21
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 classification was 67% and 85%, respectively.

Similarly, Zhang et al.[112] applied NIR-HSI (1000-1600 nm) to evaluate wheat grains 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 differenciation rates of 92.9% for

t
those contaminated by A. niger, 87.2% by A. glaucus and 99.3% by Penicillium strains.

ip
cr
Similarly another study realized by Del Fiore et al.[45] evaluated the presence of A. flavus, A.

us
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
an
used to create a model for fungal identification by Discriminant Analysis (DA). Results

revealed that between 500-700 nm the absorbance increased while fungal and mycotoxin
M
contamination increased, however, between 850-950 nm the absorbance values decreased,

especially with an A. niger strain.

ed
pt

The variation of absorbance observed in the visible spectrum (500-700 nm), could be caused by

fungi, which alter the spectral properties of maize grains related with color changes,[41] while the
ce

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]

Ac

Regarding to the chemical composition changes, Farag et al.[114] observed an increase of 0.5% in

the lipid content and between 3-9% in the protein content, as well as an increase in the reducing

sugars content up to 6 folds in wheat grains, especially those infected by A. niger. These

chemical composition changes would be the reason for the altered spectral properties in

22
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 Fusarium

verticillioides and analyzed 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 is 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]

t
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For its part, Shahin and Symons[116] used Vis/NIR-HSI (400-1000 nm) to detect Fusarium-

cr
damaged grains of wheat. LDA was applied and an overall classification accuracy ≥ 92% was

us
obtained. Later, the same authors[117] applied PLS-DA for detecting Fusarium, however a lower

overall accuracy was achieved (90%).

an
M
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
ed

(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
pt

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
ce

metabolites and changes in the chemical composition and color of the grains.[41,45,113] Also,

higher fungal growth was observed in the portion of germ, which is the fraction rich in proteins,
Ac

lipids and minerals; while the endosperm is mainly constituted by starch.[119]

23
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

t
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the means of their organic residual deposits on and around plants; while in postharvest stages

the main source is the cross contamination.

cr
us
Bacterial contamination an
In recent years, diseases caused by foodborne pathogens such as Escherichia coli, Salmonella,
M
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 route of vegetable
ed

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,
pt

early detection plays a crucial role in protecting the consumer against infectious
ce

microorganisms, so being that the HSI technology one of the best options in this regard.
Ac

Escherichia 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 analyzed 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)

24
algorithm was used to construct a prediction map of all pixel spectra of an image by coloring

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

t
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proximity of food crops and processing operations to livestock or wildlife intrusion.[124]

cr
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

us
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
an
classification accuracy was 99.5%.
M

Yang et al.[126] employed a HSI system (fluorescence mode at 320-400 nm) for the detection of
ed

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
pt

research team[127] developed and evaluated three multispectral algorithms derived from

hyperspectral line-scan fluorescence imaging (481-780 nm) for the detection of fecal
ce

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
Ac

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.

25
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

mould” (caused by Penicillium digitatum) and “blue mould” (caused by Penicillium italicum)

t
ip
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

cr
of the damaged fruit is very similar to the uncontaminated ones,[31] and therefore cannot be

us
detected in postharvest by manual inspection.
an
Mehl et al.[129] used HSI (430-900 nm) to analyze apples contaminated by fungi. An asymmetric
M
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
ed

portions of the apples examined. Also, the carotenoid absorption band (450 nm) offered good

contrast between wholesome apples with respect to samples affected by fungal contamination,
pt

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,
ce

because it requires longer data processing time, while the asymmetric second difference method

requires only three wavelengths and much less computation time to process the images. So, it
Ac

can be easily implemented in a three-band multispectral imaging (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

26
that cause spoilage in citrus fruit are included Penicillium digitatum and Penicillium italicum,

also analyzed by HSI.[131]

In this regard, a 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 optimise the

classification was estimated to be 20, selected by Genetic Algorithms based on Linear

t
ip
Discriminant Analysis (GA-LDA). The classification rate of contaminated samples, obtained by

cr
Classification and Regression Trees (CART), was above 91%, highlighting the capable of HSI

for early detection of this kind of contamination, which is extremely relevant from the

us
economical point of view.
an
More recently, Folch-Fortuny et al.[31] have developed a N-way Partial Least Squares
M
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,
ed

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
pt

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
ce

by infected fruits.
Ac

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 on-line MI system. They used a Vis/NIR-HSI system (reflectance mode

at 325-1100 nm) to discriminate between uncontaminated and decayed oranges (affected by P.

27
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

additional alternative for the citrus fruit decay detection problem. Nevertheless, despite these

good results, according to the authors, further research is still needed to establish on-line MI

t
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system for industrial applications, as well as verify performance of the proposed algorithm to

cr
detect early decay caused by other fungal species.

us
Furthermore, the most frequent signs of microbial deterioration in fruits are the reduction of
an
sugars content, elevated acidity and superficial growth of aerobic bacteria, yeasts and molds.[133]

To that respect, Teena et al.[22] evaluated the growth of Aspergillus flavus in date fruits and their
M
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),
ed

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
pt

significant wavelengths, and Linear Discriminant Analysis (LDA) and Quadratic Discriminant

Analysis (QDA) were applied in the statistical classification. Classification accuracies higher
ce

than 91% were achieved, and in the most of cases QDA yielded better accuracy in all the

models tested. However, the authors highlighted that further works are required to test this
Ac

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

28
symptoms of contamination are visible by direct observation just several days after the

infection.

Contamination by insects in plant-based foods

t
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Detection of insect damages in plant-based foods is another challenge as they can cause serious

cr
economic loss.[12,134] Non-invasive measurement is a complicated task due to the fact that larvae

us
generally hide themselves deep inside the fruits and vegetables and they cannot be easily

detected from outside.[9] In Table 3 are shown some studies carried out using HSI technology to
an
detect insect infestation in different plant based foods.
M

Xing et al.[135] developed a MI system to evaluate internal insect infestation in cherries by using
ed

of Vis/NIR-HSI in reflectance (590-1550 nm) and transmittance (580-980 nm) modes. A

Genetic Algorithm (GA) was applied to select optimal wavebands in both modes, and the best
pt

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
ce

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.
Ac

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 between wholesome and infested samples, and results showed a better accuracy in

transmittance mode (88% to 93%). The finding pointed out that the HSI system offers a better

29
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

cuased by Sitophilus oryzae, Rhyzopertha dominica, Cryptolestes ferrugineus, and Tribolium

castaneum in wheat grains. The acquired spectral data was reduced by PCA, and by using LDA

t
ip
and QDA, they correctly classified 85-100% healthy and insect-damaged wheat grains.

cr
us
Later, discrimination between insect-damaged (Etiella zinckenella Treitschke) and undamaged

vegetable soybeans was performed by Huang et al.[55] applying hyperspectral transmittance

an
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
M
accuracy of 95.6% for the validation set of samples.
ed

More recently, Mireei et al.[54] have developed a new approach to detect insect infestation in
pt

tomatoes based on a 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
ce

classify tomatoes, three different machine learning techniques: ANN, SVM and Bayesian

Networks (BN) were implemented, obtaining the best performance by ANN, based on spectral
Ac

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 hyperspectral fluorescence line-scan imaging system (400-800 nm) in order to develop a

30
simple MI algorithm to detect frass contamination at different concentrations on surfaces of

mature red tomatoes. Five wavelengths: 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 kg/L and 0.1 kg/L frass contamination spots, which is very difficult to achieve

with a simple visual inspection. However, differentiation of 0.05 kg/L and 0.02 kg/L frass

t
contamination spots was more difficult. Therefore, the efficiency of this MI system needs to be

ip
improved in future works.

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us
APPLICATIONS TO
anEVALUATE BIOLOGICAL

CONTAMINANTS IN ANIMAL-BASED FOODS

M
ed

Microbial contamination in meats and fish

pt
ce

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
Ac

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 flavors, nutrient loss, changes in appearance,

color, etc. In many cases, consumer purchasing decisions depend on meat color more than any

other quality parameters because consumers use discoloration as an indication of freshness and

wholesomeness.[6]

31
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

t
traditional microbial count methods.[141,143,144] Enterobacteriaceae is a group of pathogens

ip
including E. coli, Shigella, Salmonella or Yersinia, whose presence is used as an indicative of

cr
hygienic practices. Table 4 shows some applications of HSI for detecting microbial

us
contamination in different types of meat and marine products.
an
Microbial contamination in beef and pork meat
M

Peng et al.[74,145] evaluated microbial contamination in beef steaks applying HSI (400-1100 nm)
ed

and total viable count (TVC) methods. A Multiple Linear Regression (MLR) model was

established to predict log10 TVC, obtaining a R2p = 0.95. Besides that, they observed a relation
pt

between meat spoilage and oxidation of hemoglobin and myoglobin, which was localized in the

absorption bands of 596 and 760 nm, corresponding to the spectral signature of oxymyoglobin
ce

and oxyhemoglobin, respectively.

Ac

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, being Pseudomonas 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

32
(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.

t
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Additionally, this study revealed that reflectance decreased as the microbial population

cr
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

us
decreasing 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
an
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
M
spoilage dynamics in meat.
ed

More recently, Tsakanikas et al.[79] have employed a multispectral system in reflectance mode
pt

(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
ce

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
Ac

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 favor the growth of specific microorganisms.

33
Regarding pork, Peng and Wang[148] applied HSI (400-1000 nm) to detect bacterial

contamination, determining five optimal wavelengths by Stepwise Discrimination method: 480,

525, 650, 720, and 765 nm. Least Square Support Vector Machines (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 a R = 0.87. The proposed method

demonstrated better results than ANN or MLR methods.

t
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Likewise, Wang et al.[149] by using of a LS-SVM method obtained a R2P of 0.9426 in a study

cr
performed to predict the TVC of fresh pork meat based on HSI data. Also based on LS-SVM,

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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).
an
M
Later, Dissing et al.[151] used a multispectral imaging (MI) system in 18 different wavelengths

(405-970 nm) for spoilage degree detection of pork. In addition, a sensory evaluation panel
ed

composed of five members was recommended to judge the spoilage degree into 1 of 3 classes

(fresh, semi-fresh, and spoiled). The classification of images in these three categories, and
pt

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
ce

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
Ac

with a 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

34
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 a Linear Discriminant Analysis (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.

t
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More recently, Ma et al.[153] applied a MI system utilizing 19 different wavelengths (400-1000

cr
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 a R2 of 0.89. The prediction model

us
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
an
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.
M
ed

The versatile application of HSI would seem to offer numerous potential advantages, including

reduced labor costs, the elimination of human error and/or subjective judgment, and allowing a
pt

real-time product data creation and document generation reliable for traceability and labeling in

the meat industry.[152] However, in spite of good results, the presence of different micro flora
ce

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
Ac

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 a HSI system (scattering mode at 400-1100 nm) to determine E. coli concentration

in pork meat by the using of a MLR model, obtaining a reasonable performance with a RCV of

0.88.

35
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] Hyperspectral imaging (HSI) has been used to detect chicken meat

contamination with pathogenic microorganisms based on spectral differences between

t
ip
contaminated meat and meat from healthy chickens.[156,157]

cr
us
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
an
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-
M
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,
ed

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.
pt
ce

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
Ac

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 wavelenghts between 430-900 nm.[161]

36
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]

t
ip
A similar study was developed by Yoon et al.[158] in an online real-time HSI system (400-1000

cr
nm). The fecal detection algorithms using 517, 565 and 802 nm allowed to differentiate fecal

us
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
an
positive errors (less than 1% in some cases). Besides that, they provided a commercially viable

imaging platform for fecal detection.

M
ed

More recently, a new two band freshness index (TBFI) have used by Ye et al.[163] to develop a

bacterial prediction model (HSI vs. TVC) with a R2P of 0.6833, based on the wavelengths 650
pt

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

ce

fillets, correlating with the traditional microbiological plate count techniques.

Ac

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.

37
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

t
ip
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

cr
and 0.45 log10 CFU/g for calibration, cross-validation and prediction models, respectively. The

us
prediction map (chemical image) provided a good approach for visualizing the distribution of

Enterobacteriaceae. Moreover, an increase in spectral reflectance as the microbial load

an
increased was observed, due to the changes caused by microbial metabolism.[164]
M

Also, Feng and Sun[101] correlated the spectral information obtained by HSI (900-1700 nm) with
ed

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,

pt

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
ce

calibration, cross-validation and prediction models, respectively.

Ac

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 an increased concentrations of these substances causing undesirable odors and

tastes.[166]

38
According to these studies,[101,141,143] the full-wavelength models (HSI systems), compared to 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 on-line 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

t
ip
verify the effectiveness of HSI and to figure out the feasibility of MI systems for real-time

predicting Pseudomonas and Enterobacteriaceae loads in chicken or other meat.

cr
us
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
an
for consumers. Qin et al.[168] applied a line-scanning HSI system (500-700 nm), and two Soft

Independent Modelling by Class Analogy (SIMCA) models were developed to differentiate

M

organic residues and stainless steel samples: 2-class (“stainless steel” and “organic residue”) and

4-class (“stainless steel”, “fat”, “blood” and “feces”), whose classification accuracies were
ed

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
pt

on stainless steel surfaces), by correlation analysis.

ce
Ac

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

39
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.

Microbial contamination in fish

t
Fish and other marine products are highly perishable due to its nutritional richness, which

ip
makes them suitable for the growth of pathogenic and spoilage microorganisms. The growing

cr
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

us
entails the use of potent analysis techniques to detect possible contamination.
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Deterioration of fish can occur either as the result of autolytic endogenous enzymatic
M

degradation or by the microbial spoilage,[170] producing undesirable modifications such as

textural changes, discoloration, development of off-odors, production of slime, increasing of

ed

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
pt

such as hemoglobin and myoglobin,[175-177] and thereby generating changes in the color and
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other characteristics. In recent years, hyperspectral imaging (HSI) has been extensively studied

(Table 4) and implemented as a technological alternative to traditional analytical methods and

Ac

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-neighbor (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

40
authors reported an increment of absorbance at 636 nm, phenomenon related to the oxidation of

hemoglobin and myoglobin to methemoglobin and metmyoglobin, respectively.[146]

In the same way, Wu and Sun[144] used time series-hyperspectral imaging (TS-HSI) between

400-1700 nm to predict contamination in salmon fillets. Competitive Adaptive Reweighted

Sampling (CARS) was conducted to identify the most important wavelengths that had the

t
ip
greatest influence on the TVC prediction, and eight wavelengths: 495, 535, 550, 585, 625, 660,

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785, and 915 nm were selected. On the basis of these wavelengths, a CARS-PLSR model to

predict TVC was developed, with a R2 = 0.985 and RPD = 5.127. Similar work was carried out

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by Cheng and Sun[178] to predict TVC in carp fillets by using of HSI (400-1000 nm), observing

an increase in reflectance values. Seven optimal wavelengths: 410, 488, 553, 665, 750, 825, and
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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.
M
ed

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
pt

were also recommended to be developed for on-line applications.

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Regarding to specific microbial species, among the most important fish contaminating bacteria
Ac

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

Successive Projection Algorithm (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]

41
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 lactic acid bacteria

t
ip
(LAB) loads in salmon fillets by NIR-HIS. Eight important wavelengths: 1155, 1255, 1373,

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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

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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
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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
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wavelength spectra or six selected key wavelengths: 424, 451, 545, 567, 585, and 610 nm.
ed

In all cases, the best model was transferred to each pixel of images, and colorful distribution
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maps (chemical image) with different colors and representing different numbers of

Enterobacteriaceae, EPC or LAB counts were produced. Given the promising results, the
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simplified models (MI systems) showed better statistical indicators (R/R2, RMSE and RPD,

Table 5). Therefore, MI system developed could be more suitable for on-line applications, due
Ac

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.

42
Microbial contamination in eggs

The most studies aimed to evaluate this type of food by using HSI have been applied to inspect

t
quality attributes such as polyunsaturated fatty acids content (omega-3),[183] freshness, bubble

ip
formation or scattered yolk.[184] There is not comprehensive information available regarding the

cr
application of HSI to evaluate microbial contamination in these food products.

us
It is known that organic residues on eggshells such as blood, feathers, feces, etc. can be a
an
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
M
prevent future risk. To that respect, Lunadei et al.[186] used MI (440-940 nm) to discriminate

between clean and contaminated eggs with organic residues. They employed an image
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algorithm based on a combination of red (700 nm) and blue (450 nm) images, achieving a

classification rate near to 98% based on the geometric characteristics of the detected stains, with
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a very short processing time (0.05 seconds). Afterwards, the system was validated using a

second set of samples, obtaining a classification rate of 97%.

ce
Ac

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 on-line process. However, in spite

of the obtained results, further research is necessary in order to optimaze the accuracy, for

example, employing samples with different kinds of defects and monitoring whether changes in

the illumination system affect the results of the imaging process.

43
Evaluation of parasitic contamination

Table 4 shows some of the studies carried out by HSI application for detecting parasites in

t
different types of meat and marine products. The presence of parasites such as Anisakis simplex

ip
in fish or Trichinella spp. and Taenia solium in pork and beef, is related to the incidence of

cr
many diseases.[187-189] Parasitic infection is a severe food safety challenge, and is important for

the food industry rely on compatible preventive action to avoid the presence of parasites and on-

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line detecting methods.[64] For example, fish fillets are inspected by transillumination on

candling tables and parasites are removed manually by trained personnel,[60] achieving to
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identify up to 70% of contaminated pieces.[61] However, this procedure and other options have

disadvantages previously mentioned.

M
ed

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
pt

by hyperspectral imaging (HSI) due to the fact that their presence in fish flesh present

distinctive “spectral fingerprints” compared to normal fish muscles.[6] In this regard, a

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pioneering study was developed by Wold et al.,[77] who investigated how MI (transmittance
Ac

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.

44
Heia et al.[64] achieved a greater depth detection of nematode Gadus morhua in cod fillets using

HSI (350-950 nm). The spectral images were analyzed 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 to 3 mm deeper compared to the classic visual inspection,[60,61] besides

the method is non-intrusive and should thus be feasible for industrial purposes.

t
ip
For their part, Sivertsen et al.[190] applied HSI (400-1000 nm) to evaluate the presence of

cr
parasites in cod fillets using a mobile system at an operating speed of 25 mm/s, obtaining in

us
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
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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
M
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%
ed

(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
pt

of detection of parasites and the correct detection rate in future applications.

ce

It is also reported the possible use of HSI to evaluate the presence of non-pathogenic parasites
Ac

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

45
transmittance between 600-950 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, whilst there is not

comprehensive information available in the bibliography regarding the application of HSI in

t
ip
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

cr
discriminate between both infected and non-infected meat by using Soft Independent Modeling

us
of Class Analogy (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
an
be considered as the best muscle for parasite identification purposes in contaminated pork.

Finally, it should be noted that there is not information available in the bibliography regarding
M

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
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undercooked pork consumption.[187,194]

pt

CURRENT AND FUTURE CHALLENGES

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Advantages and disadvantages of hyperspectral imaging (HSI)

Ac

This review gives special prominence to hyperspectral imaging (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 cannot

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

46
potential to realize quantitative and qualitative analysis as well as rapid real-time and on-line

detection,[11] aspects that with other technologies is not always possible.

However, HSI technology still has some disadvantages that need to be resolved for further

application 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 to

t
ip
chemical-based analytical methods.[11] Moreover, hyperspectral images contain much

cr
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

us
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 on-line purpose.[5]
an
M
To overcome these disadvantages, current studies are aiming to develop models that allow (a) a

rapid data discrimination and retrieve interesting information, given the large amount of data
ed

from the wide range of spectral bands in which the HSI works, and (b) identifying optimal

wavelengths for each food or food constituents, affected by biological contaminants. This is
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possible thanks to the potential of HSI to identify and discriminate between different

constituents,[76] biological contaminants[110] or metabolites produced by contaminant

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microorganisms, such as toxins[42,45,100,106] or other substances causing undesirable attributes in

food products.[109,172-174]
Ac

47
FUTURE CHALLENGES

On the basis of aforementioned disadvantages, scientific and technological challenges that must

t
be overcome for further application of HSI at the industrial level are:

ip
a. Further research for detecting biological contaminants in liquid foods is needed,[15]

cr
since most applications have been focused on solid samples (Tables 2-4).

us
b. Quantification of low concentrations of microorganisms, since most studies have been

focused on assessing microbial densities higher than 102 CFU/g.[2]

an
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
M
contaminants.

d. Advanced methodologies that allow discrimination between biological contaminants

ed

based on its specific "spectral signature".

e. Optimize the existing models or create more robust models that allow to predict
pt

biological contamination from the spectral data obtained, without running the risk of

losing valuable information.

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f. Validation of developed models on several storage conditions, simulating real life, for

example at different storage temperatures.[79]

Ac

g. The design of techniques able to fuse the spatial and spectral data, since most of

available hyperspectral data processing techniques focus on analyzing the spectral data

without incorporating information on the spatial data.[3]

Besides, the implementation of on-line applications, due to the big size of hyperspectral images,

that requires efficient technologic tools to analyze such images in a real-time mode. The most

hyperspectral raw data is currently used in off-line systems at laboratory level to select key

48
wavelengths for building multispectral imaging (MI) systems suitable for on-line

applications[6,10,14]. Therefore, 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

t
instrument cost and high analytical speed.[8,9]

ip
cr
Compared with the full-wavelength models (HSI systems), the simplified models (MI systems)

us
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
an
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
M
also be part of the reasons for model enhancement.[14]
ed

Likewise, some disadvantages in analysis of microorganisms concerning food quality could be

pt

overcome with the technical integration of HSI with fluorescence microscopic imaging (FMI)

and Raman microscopic imaging (RMI), improving the capability for inspection of bacterial
ce

contamination. 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
Ac

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

49
by identifying specific spectral bands. Heia et al.[64] detected parasites at a depth of 8 mm at

wavelengths between 350-950 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 approximately 1 μm, limiting its application to

surface analysis.[195] Additionally, according to previous results obtained by Sivertsen et

t
al.,[62,190] transmittance mode allowed to obtain better results in detecting parasites, as well as the

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interactance mode, which has less surface effects and reduces the influence of thickness.[12]

cr
us
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
an
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
M
imply to build different systems incorporating different wavelengths depending on the

variety.[31] Finally, regarding to high cost of the HSI systems compared with other technologies,
ed

this is expected to be a minor barrier in the next years because of the increase of commercial

suppliers could reduce the cost and improve its availability.[6] In the last years, commercial
pt

HSI/MI systems for food applications such as Hyperspec Inspector (Headwall Photonics,

Fitchburg, United States) or SisuCHEMA (Specim, Oulu, Finland) are appearing on the
ce

market,[10] and recently, Goel et al.[196] have developed a prototype capable of capturing
Ac

hyperspectral images with a low-cost camera (called HyperCam), which uses 17 wavelengths

that are spread between 450-990 nm, with a cost of about US$ 800, but could reach US$ 50 to

fit on mobile phones.

50
CRITICAL APPRECIATION AND CONCLUSIONS

Of the numerous applications of hyperspectral imaging (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

t
ip
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

cr
origin, the industrial processing, packaging and the storage conditions.

us
an
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
M
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
ed

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,
pt

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 as feces, digestive
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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
Ac

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

51
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, technological 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 applications as an

alternative to traditional techniques such as the liquid chromatography, the MID-FTIR

t
spectroscopy or the ELISA and PCR tests, which besides being tedious, are expensive and their

ip
application is limited on the laboratory level. Finally, although many researches for evaluating

cr
biological contamination in foods has been performed, much studies are still needed to

standardize the procedure of HSI in terms of data mining, MI development and further real-time

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and on-line applications along the various stages in the food production chain.
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FUNDING

This study was funded by the following institutions: Programa Nacional de Innovación para la
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Competitividad y Productividad Innóvate Perú – Ex-FINCyT (Contract 407-PNICP-PIAP-2014)

and Universidad Nacional de Trujillo - UNT (PIC2-2013/UNT).

ed
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Ac

52
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and control: A review. Critical Reviews in Food Science and Nutrition 2012, 52(11),
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2. Gowen, A.A.; Feng, Y.; Gaston, E.; Valdramidis, V. Recent applications of

hyperspectral imaging in microbiology. Talanta 2015, 137, 43-54.

t
3. Liu, D.; Zeng, X.A.; Sun, D.W. Recent developments and applications of hyperspectral

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imaging for quality evaluation of agricultural products: A review. Critical Reviews in
Food Science and Nutrition 2015, 55(12), 1744-1757.

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Figure 1 Main parameters controlled in food production

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ed
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Figure 2 Schematic of the hyperspectral imaging (HSI) system: a Acquisition of hyperspectral
image. b Hypercube: Spectral and spacial information in a hyperspectral image at different
wavelengths. c Spectral signature for each constituent of the sample plotted against different
wavelengths

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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]

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Total bacteria and PPC Pork [152]

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LAB Salmon [172]
Pseudomonas, Brochothrix Different meats [101,141,142,180]

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thermosphacta and
Enterobacteriaceae
Microscopy Bacteria and fungi Different foods [21]

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ELISA Fungal toxins Cereals [37,39]
Bacterial toxins Meat, sausage, milk and [38,40]
mayonnaise
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Virus Crops [89,90]
Anisakis simplex proteins Sea products [67,71]
Trichinella Pork [68]
M
Taenia saginata Beef and pork [69,70]
PCR Virus Crops [90]
Salmonella Meats and milk [197]
ed

Bacteria Fruit crops [198]

Fungal toxins Cereals [199]
Taenia saginata Beef and pork [66,70]
HPLC and TLC Fungal toxins Cereals [41-43]
pt

LC-MS Anisakis simplex proteins Fish [72]

Detection by Fungal toxins Cereals [43,48-50]
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fluorescence
Ultrasound LAB, Aspergillus niger and Fruit juices [30]
Saccharomyces cerevisiae
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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]
Abbreviations: 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.

69
Table 2 Spectral imaging applications for detecting microbial contamination in agricultural
crops

Cultivar Contaminating agent Aim λ (nm) Reference

Maize - Fungi: Helminthosporium maydis Spectral differentiation in 400-2700 [81]

the NIR region (800-2600
- Virus: MDMV nm) between healthy,
MDMV infected, and H.
maydis-infected leaves

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Wheat Fungi: Fusarium culmorum Correct classification 400-1000 [84]
between diseased and

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healthy ear tissues

Strawberry Fungi: Colletotrichum gloeosporioides Early detection of 400-1000 [80]

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infection stages of
strawberry foliar
anthracnose

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Citrus fruit Bacteria: Xanthomonas axonopodis Multispectral algorithms 450-930 [85]
to differentiate “citrus
canker” from other peel
conditions
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Grapevine Virus: GLRaV-3 Evaluation of the 400-1000 [92]
reliability of hyperspectral
airborne imaging to
remotely detect GLRaV-3
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vineyards

Correct classification 350-2500 [93]

between infected from
healthy vine leaves
ed

Watermelon Virus: CGMMV To discriminate virus- 946-2016 [94]

seeds infected watermelon seeds
from healthy ones
pt

Abbreviations: MDMV: maize dwarf mosaic virus; GLRaV-3: grapevine leafroll-associated virus-3; CGMMV:
cucumber green mottle mosaic virus.
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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 Mode a λ (nm) b Accuracy c Reference

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]

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Maize T 500-950 95% [102]

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2
Fumonisin Milled maize R 720-940 R = 0.68 [42]
Ochratoxin A Barley R 1000-1600 100% [98]

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Viable microorganisms and diseases
Aspergillus and Fusarium Maize R 400-1000 97.0, 94.5% [45]
Aspergillus and Penicillium Barley R 1000-1600 100% [98]

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verrucosum
Fusarium Maize A 1000-2498 R2 = 0.98 [100]
Aspergillus niger Wheat R 1000-1600 92.9% [112]
Aspergillus glaucus Wheat
an R 1000-1600 87.2% [112]
Penicillium Wheat R 1000-1600 99.3% [112]
2
Aspergillus orizae Rice R 400-1000 R = 0.97 [118]
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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]
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R 730 y 830 95.3% [87]

Fecal contamination Apple F and R 400-1000 100 and 99.5% [125]
Apple F 480-780 99% [127]
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2
Escherichia coli Spinach R 400-1000 R = 0.97 [20]
Frass Tomatoe F 400–800 > 99% [138]
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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]
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Callosobruchus maculatus Mung bean R 1000-1600 82-85% [200]

Etiella zinckenella Soybean T 400-1000 95.6% [55]
Treitschke
a
A: Absorbance. F: fluorescence. R: reflectance. T: transmittance
b
Wavelenght
c
R2: coefficient of determination

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 Table 4 Hyperspectral imaging (HSI) for detecting viable microorganisms and parasites in meat
and marine products

Contaminant a Food Mode b λ (nm) c Accuracy d Reference

Fecal material and Poultry carcasses R 400–1000 89-98% [158]
ingesta
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]

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Pseudomona Chicken meat R 910–1700 R = 0.87 [101]
2
Enterobacteriaceae Chicken meat R 900–1700 R = 0.87 [141]
Salmon R 900-1700 R = 0.95 [181]

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LAB Salmon R 900-1700 R = 0.940, 0.925 [172]
EPC Salmon R 900-1700 R = 0.964, 0.953 [180]

us
TVC Chicken meat R 900–1700 R = 0.94 [143]
2
Chicken meat R 400–1000 R = 0.6833 [163]
2
Pork R 400–1100 R = 0.987, 0.942 [149]
Pork
an R 400–1100 2
R = 0.9236 [150]
2
Pork R 900–1700 R = 0.82 [152]
Pork R 400–1100 R = 0.886, 0.863 [154]
2
Beef S 400–1100 R = 0.95 [74]
M
Beef R 405-970 R2 = 0.98 [79]
2
Beef S 400–1100 R = 0.96 [145]
2
Salmon R 400–1700 R = 0.985 [144]
ed

2
Carp R 400-1000 R = 0.90 [178]
TBC Salmon I 400-1000 88% [170]
Parasites Cod I 400–1000 60.3-70.8% [62]
pt

Cod T 400–1000 58% [190]

Clam T 400-1000 85% [192]
a
LAB: Lactic acid bacteria. EPC: Enterobacteriaceae and Pseudomonas count. TVC: total viable count. TBC: total
ce

bacterial count
b
R: reflectance. S: scattering. T: transmittance. I: interactance. F: fluorescence.
c
Wavelenght
Ac

d
R: correlation coefficient. R2: coefficient of determination

72
Table 5 Multispectral imaging (MI) models developed from hyperspectral imaging (HSI)
models by selecting of key wavelengths

HSI (full-wavelengths) MI (optimized model)

Food sample: Statistical Selected Statistical
λ (nm) Reference
contaminant indicators wavelengths indicators

Wheat grains: 400-1000 nm Calibration 4 wavelengths: Calibration model: [32]

t
2 2
mildew damage model: R = 450, 561, 861, R = 0.89,

ip
C C

0.89, RMSEC = and 917 nm RMSEC = 0.68

0.67

cr
Validation model:
Validation model: R2V = 0.87,
R2V = 0.87, RMSEV = 0.74

us
RMSEV = 0.73

Chicken fillets: 900-1700 nm Calibration 14 wavebands in Calibration model: [101]

Pseudomona loads
an
model: R = 0.88, 5 spectral R = 0.91, RMSE =
RMSE = 0.60 segments: 1138- 0.55 log10 CFU/g
log10 CFU/g 1155, 1195-
Cross-validation
1198, 1392-
M
Cross-validation model: R = 0.87,
1395, 1452-
model: R = 0.83, RMSE = 0.65
1455 and 1525-
RMSE = 0.71 log10 CFU/g
1529 nm
ed

log10 CFU/g
Prediction model:
Prediction model: R = 0.88, RMSE =
R = 0.81, RMSE 0.64 log10 CFU/g
pt

= 0.80 log10
CFU/g
ce

Chicken fillets: 930-1450 Calibration 3 wavelengths: Calibration model: [141]

2 2
Enterobacteriaceae model: R = 0.88, 930, 1121, and R = 0.89, RMSE
loads RMSE = 0.35 1345 nm = 0.33 log10
Ac

log10 CFU/g CFU/g

Cross-validation Cross-validation
2
model: R = 0.82, model: R2 = 0.86,
RMSE = 0.45 RMSE = 0.40
log10 CFU/g log10 CFU/g

Prediction model: Prediction model:

2
R = 0.85, RMSE R2 = 0.87, RMSE
= 0.47 log10 = 0.45 log10

73
 CFU/g CFU/g

Chicken fillets: TVC 910-1700 nm Calibration 10 wavelengths: Calibration model: [143]

model: RC = 0.97, 961, 1054, 1081, RC = 0.96, RMSEC
RMSEC = 0.37 1084, 1191, = 0.42 log10
log10 CFU/g 1198, 1201, CFU/g
1208, 1218, and
Cross-validation Cross-validation
1328 nm
model: RCV = model: RCV =
0.93, RMSECV = 0.93, RMSECV =

t
ip
0.57 log10 CFU/g, 0.54 log10 CFU/g,
RPD = 2.60 RPD = 2.75

cr
Salmon: LAB 900-1700 nm RP = 0.929, 8 wavelengths: RP = 0.925, [172]
RMSEP = 0.515 1155, 1255, RMSEP = 0.531
log10 CFU/g 1373, 1376, log10 CFU/g

us
1436, 1641,
1665, and 1689
nm
an
Salmon flesh: 900-1700 nm RP = 0.954, RPD 9 wavelengths: RP = 0.964, RPD = [180]
Enterobacteriaceae = 3.313, RMSEP 931, 1138, 1175, 3.715, RMSEP =
M
loads = 0.481 log10 1242, 1359, 0.429 log10 CFU/g
CFU/g 1628, 1641,
1652, and 1655
nm
ed

Salmon flesh: 900-1700 nm RP = 0.94, RPD = 8 wavelengths: RP = 0.95, RPD = [181]

Enterobacteriaceae 2.97, RMSEP = 924, 931, 964, 3.23, RMSEP =
pt

loads 0.53 log10 CFU/g 1068, 1262, 0.47 log10 CFU/g

1373, 1628, and
1668 nm
ce

Carp: Escherichia 400-1000 nm R2P = 0.88, 6 wavelengths: R2P = 0.87, [182]

coli loads RMSEP = 0.26 424, 451, 545, RMSEP = 0.27
Ac

log10 CFU/g, RPD 567, 585, and log10 CFU/g, RPD

= 5.47 610 nm = 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 (RP/R2P, RMSEP), respectively.
RPD: residual predictive deviation.

74