toxins

Review
E-Nose Technology for Mycotoxin Detection in Feed:
Ready for a Real Context in Field Application or Still
an Emerging Technology?
Federica Cheli 1,2, * , Matteo Ottoboni 1 , Francesca Fumagalli 1 , Sharon Mazzoleni 1 , Luca Ferrari 1
and Luciano Pinotti 1,2

1 Department of Veterinary Medicine and Animal Science, University of Milan, 26900 Lodi, Italy
2 CRC I-WE (Coordinating Research Centre: Innovation for Well-Being and Environment), University of Milan,
20100 Milan, Italy
* Correspondence: [email protected]

Abstract: Mycotoxin risk in the feed supply chain poses a concern to animal and human health,
economy, and international trade of agri-food commodities. Mycotoxin contamination in feed and
food is unavoidable and unpredictable. Therefore, monitoring and control are the critical points.
Effective and rapid methods for mycotoxin detection, at the levels set by the regulations, are needed
for an efficient mycotoxin management. This review provides an overview of the use of the electronic
nose (e-nose) as an effective tool for rapid mycotoxin detection and management of the mycotoxin
risk at feed business level. E-nose has a high discrimination accuracy between non-contaminated
and single-mycotoxin-contaminated grain. However, the predictive accuracy of e-nose is still limited
and unsuitable for in-field application, where mycotoxin co-contamination occurs. Further research
needs to be focused on the sensor materials, data analysis, pattern recognition systems, and a better
understanding of the needs of the feed industry for a safety and quality management of the feed
supply chain. A universal e-nose for mycotoxin detection is not realistic; a unique e-nose must be
designed for each specific application. Robust and suitable e-nose method and advancements in
signal processing algorithms must be validated for specific needs.

Citation: Cheli, F.; Ottoboni, M.; Keywords: feed safety; mycotoxins; electronic nose
Fumagalli, F.; Mazzoleni, S.;
Ferrari, L.; Pinotti, L. E-Nose Key Contribution: E-nose represents a powerful tool in the feed chain as a rapid and cost-effective di-
Technology for Mycotoxin Detection agnostic tool for a rapid detection of mycotoxin contamination. Before e-nose can move from research
in Feed: Ready for a Real Context in into the feed industry, several challenges must be overcome to improve e-nose performance. Further
Field Application or Still an research is needed on e-nose technology, such as sensor materials, data analysis, pattern recognition
Emerging Technology? Toxins 2023,
systems, and on the specific needs of the feed industry for a safety and quality management of the
15, 146. https://doi.org/10.3390/
feed supply chain.
toxins15020146

Received: 9 December 2022

Revised: 17 January 2023
Accepted: 4 February 2023 1. Introduction
Published: 11 February 2023
Mycotoxins are one of the largest safety risks for the feed/food chain, with a negative
impact on animal and human health, economy, and international trade of feed and food
commodities [1–6]. Despite the availability of several strategies for prevention and control
Copyright: © 2023 by the authors.
of fungal contamination, mycotoxin contamination in feed and food is unavoidable and
Licensee MDPI, Basel, Switzerland. unpredictable [6–8]. The challenge is to minimize the effects. The global trade of agricultural
This article is an open access article commodities, the climate change scenario, and the lack of harmonization in mycotoxin
distributed under the terms and regulation are the main topics underlying the need of tools for the feed industry to manage
conditions of the Creative Commons the mycotoxin risk. It is undeniable that mycotoxin management demands an integrated
Attribution (CC BY) license (https:// approach using proactive, innovative, and improved strategic actions all along the feed
creativecommons.org/licenses/by/ chain [9]. Moving from science to practice, the first need is the availability of rapid and
4.0/). on-site analytical methods. At the feed industry level, notwithstanding the availability

Toxins 2023, 15, 146. https://doi.org/10.3390/toxins15020146 https://www.mdpi.com/journal/toxins

Toxins 2023, 15, 146 2 of 17

of advanced methods, there is a need for effective and rapid analytical methods for feed
mycotoxin detection at the levels that are set by the regulations for an efficient mycotoxin
risk management. Mycotoxins are regulated worldwide, but the set maximum levels vary
greatly from country to country [10,11]. The European Union (EU) harmonized regulations
on maximum levels of mycotoxins in feed among its member states [12–14]. A rapid, low-
cost, high-throughput analytical approach for mycotoxin detection is a need at the industry
level to make rapid management decisions on the acceptance or rejection of a lot [6]. The
need for rapid methods and criteria to be considered for validation of methods to be used
for mycotoxin detection were topics discussed in a “Special Issue: Rapid methods for
mycotoxin detection” and “Special Issue: Rapid Detection of Mycotoxin Contamination”
published in World Mycotoxin Journal and Toxins, respectively [15,16]. Within rapid
methods, electronic nose (e-nose) may represent an attractive and promising method for
mycotoxin detection.
After a brief survey on mycotoxin contamination in animal feed, this review provides
an overview of the use of e-nose as an effective tool for rapid mycotoxin detection and
management of the mycotoxin risk at feed business level.

2. Mycotoxin Contamination
Mycotoxins are secondary fungal metabolites. Fusarium, Aspergillus, Penicillium, and
Claviceps spp. mycotoxins produced by represent the main contaminants of the feed supply
chain, with important impact on animal health, productivity, and feed/food safety [5]. Of
the more than 300 mycotoxins identified up to now, aflatoxins (AFs), aflatoxin B1 (AFB1 ),
deoxynivalenol (DON), zearalenone (ZEA), fumonisins B1 and B2 (FBs, FB1 , and FB2 ),
ochratoxin A (OTA), T2, and H-T2 are regulated by EU legislation for animal feed [12–14].
Mycotoxin contamination occurs in feed all along the feed supply chain, including
production, processing, storage, and distribution. Extensive surveys were carried out on
mycotoxin occurrence in feed raw materials and complete feeds. However, forages must
also be monitored because of their significant contribution to total mycotoxin intake [17].
Feed contamination may also represent a safety risk for humans because of the possible
carry-over of mycotoxins into animal-derived food [18–21]. The main complete feed and
feed raw materials analysed worldwide for mycotoxin contamination were grains and grain
co-products (bran, corn gluten meal, dried distillers’ grains, and solubles). Less data are
available for other feed ingredients, such as soybean meal, cotton seed, sorghum, cassava,
peanut, and copra. [8,21–35].
Several important findings resulted from these multiannual mycotoxin surveys in
animal feed. The overall results confirm that AFs, DON, FBs, OTA, T-2, and HT-2 toxins
and ZEA are the main mycotoxins occurring in feed and are invariably found in cereal
grains. Moreover, although the incidence of samples contaminated with mycotoxins above
the EU legislative limit or recommended levels is low, there is a high variability, and several
samples can exceed the levels. This confirms the need for a continual monitoring activity to
check feed safety. Considerable differences in the mycotoxin profile (type and prevalence
of mycotoxin contamination) in different geographic regions of the world and year by year
variations have been reported [6,26,27,29–31]. Climatic and weather conditions (excessive
moisture, temperature extremes, humidity, and drought) during critical plant growing
stages, as well insect damage, crop systems, and some agronomic practices can cause
plant stress and determine the severity of mycotoxin contamination [5,36–38]. In this
scenario, climate change may have significant implications and effects on the distribution
and occurrence of mycotoxins in the agri-food chain [39–42].
The second finding is that co-occurrence of mycotoxins is the norm not the exception.
Multi-mycotoxin contamination was more prevalent in feed samples from Asia (82%)
than from Europe and America (40%) [6]. The most frequently co-occurring mycotoxin
combinations in compound feed were DON and ZEA; DON, T-2, and HT-2; ZEA, T-2, and
HT-2; and DON, T-2, HT-2, and ZEA. Quite high co-occurrence level was found for OTA in
combination with DON, T-2, and HT-2 [8,21,26,27,29,31,33,34].
Toxins 2023, 15, 146 3 of 17

Concerns about the safety of contaminated products have been further heightened
by modified and emerging mycotoxin. As reported by several authors [43,44], the anal-
ysis of the mycotoxin content of samples containing these compounds can lead to their
underestimation. The same author highlighted that such bias in masked mycotoxin de-
tection might be due to several analytical issues. This implies that modified mycotoxins
are hardly detected by routine analysis. This emerging issue was accessed by EFSA in a
Scientific Opinion [45] on the risks for human and animal health related to the presence of
modified forms of certain mycotoxins in food and feed. In the present opinion, all modified
mycotoxins produced by plant or fungi metabolism, formed during feed/food process-
ing, and resulting from the carry-over from contaminated feed are considered. Despite
the increasing attention paid to modified mycotoxins, data on the formation, occurrence,
toxicity, metabolic dynamics, and specific analytical methods are still rather scarce. Results
from multiannual mycotoxin surveys in feed materials and complete feed indicate the
presence of non-regulated mycotoxins: co-contamination of modified and emerging myco-
toxins with regulated mycotoxins were reported [29,32]. Currently, worldwide legislation
considers only mycotoxin mono-exposure data and does not address relevant mycotoxin co-
contamination. Moreover, recently modified and emerging mycotoxins have been included
in the EFSA risk assessments [46]. The impact of relevant mycotoxin combinations, regu-
lated and not regulated mycotoxins, should be considered, and legislation must consider
this topic in the near future.

3. Mycotoxin Analysis
The starting point of an effective mycotoxin analysis is sampling. This is a critical issue
to obtain reliable results [47]. Research on this topic continues to evolve; however, sampling
and sampling procedure are not the topic of this paper. For those who are interested, several
papers are available for further and recent information [48–52]. Regarding sampling, recent
publications on sampling techniques for grain dust and for pooling samples for mycotoxin
screening could have a huge impact for the feed industry [53,54].
The official controls of feed and food are regulated by the Regulation (EU) 2017/625,
Commission Regulation (EC) 152/2009, and Commission Regulation (EC) No 401/2006,
laying down the methods of sampling and analysis for the official control of the levels
of mycotoxins in feed and food, respectively [55–57]. These Regulations provide precise
details regarding the methods of sampling, acceptance parameters, criteria for sample
preparation, analytical performance criteria of the methods of analysis, and criteria for
reporting and interpretation of the results. Identification criteria for mycotoxin limit
of quantification (LOQ) have been the focus of a guidance document released by the
European Commission [58]. At research levels, there is continual work to develop and
validate methods for mycotoxin determination. Chromatography with MS/MS is the
reference method for mycotoxin analysis in regulated matrices and is almost routinely
performed. Studies regarding the implementation of LC-MS/MS methods, application
of chromatography with targeted and non-targeted high-resolution mass spectrometry
(HRMS), and optimisation of sample preparation for multi-mycotoxin analysis, including
modified mycotoxins, have been reported in recent years [59,60].
At the feed industry level, the on-site quality and safety of products need to be
continually monitored, and the adoption of a rapid, low-cost, high-throughput screening
methods is a must for the management of mycotoxin risk [61]. Commercially available
enzyme-linked immunosorbent assays (ELISA) kits are widely used due to their relatively
low cost and easy application. ELISA assays meet the industrial needs in monitoring and
surveillance programs as a “fit-for-purpose” tool. The development and validation of new
ELISA and lateral flow immunoassays (LFIA) methods are still an area of great interest,
including research on miniaturisation and multiplex new biosensors [59,60]. Among
traditional method for mycotoxin detection, thin-layer chromatography (TLC) is considered
to be an effective screening method for mycotoxins [62]. This traditional method has gained
great significance as a simple, rapid, and economical method for quantitative detection,
Toxins 2023, 15, x FOR PEER REVIEW 4 of 17

including research on miniaturisation and multiplex new biosensors [59,60]. Among tra-
Toxins 2023, 15, 146 ditional method for mycotoxin detection, thin-layer chromatography (TLC) is considered 4 of 17
to be an effective screening method for mycotoxins [62]. This traditional method has
gained great significance as a simple, rapid, and economical method for quantitative de-
tection, but the poor accuracy and low sensitivity make quantification difficult. This
but the poor accuracy and low sensitivity make quantification difficult. This method is
method is particularly effective in AFs and OTA determination [63].
particularly effective in AFs and OTA determination [63].
In addition to conventional analytical methods, several authors recently evaluated
In addition to conventional analytical methods, several authors recently evaluated
electrochemical aptasensors for mycotoxin detection. Ong and co-workers [64] summa-
electrochemical aptasensors for mycotoxin detection. Ong and co-workers [64] summarized,
rized, in a recent study, most recent advances in conventional methods and electrochem-
in a recent study, most recent advances in conventional methods and electrochemical
ical aptasensors for mycotoxin detection. Considering this innovative technology, its main
aptasensors for mycotoxin detection. Considering this innovative technology, its main
advantages are related to flexible modification of functional groups, high sensitivity, wide
advantages are related to flexible modification of functional groups, high sensitivity, wide
detection range
detection range of
of mycotoxin
mycotoxin types
types due
due to
to its
its flexibility
flexibility in
in electrode
electrode surface
surface modification,
modification,
simple operating
simple operating procedure,
procedure, and
and low
low cost
cost ofoffabrication.
fabrication. The
The main
main disadvantage
disadvantage is is the
the
need for surface modification and signal amplification for a high
need for surface modification and signal amplification for a high sensitivity. sensitivity.
ItIt is
is well
well known
knownthat
thatfungal
fungalspoilage
spoilageis is
responsible
responsibleof organoleptic deterioration
of organoleptic and
deterioration
off-flavour production associated with mycotoxins production [65,66].
and off-flavour production associated with mycotoxins production [65,66]. Therefore, Therefore, within
rapid methods,
within e-nose, e-nose,
rapid methods, capablecapable
of recognizing simple orsimple
of recognizing complex odours, could
or complex represent
odours, could
a fast and accurate tool in feed safety assessment by farmers and feed industry
represent a fast and accurate tool in feed safety assessment by farmers and feed industry for myco-
toxin
for screening.
mycotoxin screening.

4. Electronic
4. Electronic Nose
Nose
Ane-nose
An e-noseconsists
consistsof of an
an array
array of
of non-specific
non-specific chemical
chemical sensors
sensors with
with partial
partial specificity
specificity
and an
and an appropriate
appropriate pattern-recognition
pattern-recognition system
system thatthat can
can recognize
recognize simple
simple or or complex
complex
odours
odours[67].
[67].Sensors
Sensorsinteract
interactwith
withdifferent
differentvolatile
volatile organic
organiccompounds
compounds (VOCs),
(VOCs), providing
provid-
signals thatthat
ing signals can can
be utilized effectively
be utilized as as
effectively a unique
a unique flavour
flavourfingerprint
fingerprintofofa aproduct.
product.TheThe
application
applicationofofaarobust
robustpattern
patternrecognition
recognitionsystem
systemmakes
makespossible
possiblethe
theidentification
identificationand/or
and/or
quantification
quantification ofof the
the odours
odours [68,69].
[68,69]. The
The workflow
workflow of of an
an e-nose
e-nose analysis
analysis is is reported
reported in
in
Figure
Figure1.1.

Figure1.1. Analytical
Figure Analytical workflow
workflowfor
fore-nose
e-noseanalysis.
analysis.

There
Thereare
areseveral
severalsensor
sensordevices
devices forfor e-nose
e-nose using
using different
different types
types of
of detection:
detection: optical,
optical,
thermal,
thermal, electrochemical, and gravimetric [70,71]. Within these types ofsensors,
electrochemical, and gravimetric [70,71]. Within these types of sensors, the
the most
most
popular
populare-nose
e-nosesensors
sensorsare
aremetal–oxide
metal–oxidesemiconductor
semiconductor (MOS),
(MOS), metal–oxide
metal–oxide semiconductor
semiconduc-
field-effect transistors
tor field-effect (MOSFET),
transistors (MOSFET), andandconducting
conducting polymer
polymer (CP) and
(CP) piezoelectric
and piezoelectriccrystal
crys-
sensors. The different sensor technologies affect their performance, such
tal sensors. The different sensor technologies affect their performance, such as response as response
and
and recovery times, sensitivity,
recovery times, sensitivity, detection
detectionrange,
range,operating
operatinglimitations,
limitations,andand inactivation
inactivation by
by poisoning agents. Gas molecules interact with sensors by absorption,
poisoning agents. Gas molecules interact with sensors by absorption, adsorption, or chem- adsorption, or
chemical reactions.
ical reactions. According
According to thetotype
the of
type of sensors,
sensors, this reaction
this reaction causescauses a modification
a modification of the
of the sensor resistance, electrical conductivity, or resonance frequency, and
sensor resistance, electrical conductivity, or resonance frequency, and these changes are these changes
are measured
measured as electrical
as an an electrical signal
signal producing
producing a fingerprint
a fingerprint of VOCs.
of VOCs. There There
was anwas an
instru-
instrumental evolution, leading to a wide diffusion of commercially
mental evolution, leading to a wide diffusion of commercially available e-noses, auto- available e-noses,
automated, hybrid instruments with a combination of different sensor technologies, small
mated, hybrid instruments with a combination of different sensor technologies, small size,
size, and portable e-noses [72,73]. A universal e-nose, coping with every odour profile,
and portable e-noses [72,73]. A universal e-nose, coping with every odour profile, is not
is not realistic, and unique e-noses must be specifically designed and set up, and data
processing must be validated for specific research work. Despite their different mechanisms,
most of the sensors interact non-selectively with volatile molecules showing non-specific
recognition. The result is a “fingerprint” of the VOCs. An instrumental evolution of
e-nose is represented by a new generation of e-nose instruments based on ultra-fast gas
 realistic, and unique e-noses must be specifically designed and set up, and data processing
must be validated for specific research work. Despite their different mechanisms, most of
Toxins 2023, 15, 146 the sensors interact non-selectively with volatile molecules showing non-specific recogni- 5 of 17

tion. The result is a “fingerprint” of the VOCs. An instrumental evolution of e-nose is

represented by a new generation of e-nose instruments based on ultra-fast gas chroma-
tography. They share
chromatography. Theythe fast-screening
share capability
the fast-screening of other
capability oftypes
other of e-noses,
types while allow-
of e-noses, while
allowing,
ing, at theat same
the same time,
time, specific
specific identification
identification andquantification
and quantificationof of the
the detected volatile
volatile
molecules. Applicationsofofultra-fast
molecules. Applications ultra-fastGC GCelectronic
electronic nose
nose areare reported
reported forfor
foodfood safety
safety au-
authentication
thentication and and adulteration
adulteration analysis
analysis [74–76].
[74–76].
Data
Data analysis
analysis and
and pattern
pattern recognition
recognition (PARC)
(PARC) areare fundamental
fundamental parts parts of
of the
the e-nose
e-nose
analysis.
analysis. E-nose analysis generates a great volume of data that requires the applicationof
E-nose analysis generates a great volume of data that requires the application of
multivariate
multivariate methods
methods forfor data
data analysis.
analysis. There
There are
are aa variety
variety ofof PARC
PARCmethods
methodsthat thatcan
canbe be
used
useddepending
dependingon onthe
thetype
typeofof
data
dataand thethe
and required
requiredresults (Figure
results 2). For
(Figure a comprehensive
2). For a comprehen-
description and discussion
sive description and discussionregarding analysis
regarding of e-nose
analysis data,data,
of e-nose readers are referred
readers to the
are referred to
literature [69,77–79].
the literature [69,77–79].

Figure2.2.Overview
Figure Overviewofofthe
themost
mostcommonly
commonly used
used multivariate
multivariate pattern
pattern analysis
analysis methods
methods of e-nose
of e-nose data.
data.
Applications of e-nose analysis range from the feed/food industry and medical in-
dustry Applications of e-nose
to environmental analysis range
monitoring from the
and process feed/food
control industry
[80–83]. andapplications
The first medical in-
dustry to environmental monitoring and process control [80–83]. The
of e-nose for food analysis date to the beginning of the 1990s [84,85]. At research and first applications of
e-nose for industry
feed/food food analysis
levels,date to technology
e-nose the beginning has of
beentheemployed
1990s [84,85]. At research
for quality controlandof
feed/foodprocess,
products: industryfreshness,
levels, e-nose technology
and maturity has beenshelf-life
monitoring, employed for quality authentic-
investigations, control of
products:
ity process,
assessments, foodfreshness, and maturity
fermentation monitoring,
process, animal sourceshelf-life investigations,
food, microbial pathogen,authen-
and
ticity assessments,
pesticide food fermentation
detection [69,72,86–89]. From the process, animal source
first applications food,
of the microbial
analysis pathogen,
with the e-nose,
and pesticide
there have beendetection
no major[69,72,86–89].
changes in the From the firstfields,
application applications of thedifferences
while many analysis withcan the
be
found
e-nose,atthere
the level
haveofbeen
instrumental properties,
no major changes data
in the collection,fields,
application and processing
while many processes.
differences
can be found at the level of instrumental properties, data collection, and processing pro-
5. Volatilome: VOCs Associated with Fungal Metabolism
cesses.
Volatile compounds are related to feed and food quality, aromatic attributes, and pleasant
or
5. unpleasant
Volatilome:smell.
VOCsVolatile compounds
Associated are a group
with Fungal of carbon-based chemicals with low
Metabolism
molecular weight and high vapor pressure produced
Volatile compounds are related to feed and food by bacteria
quality,andaromatic
fungi as aattributes,
result of their
and
metabolism,
pleasant or and numerous
unpleasant of VOCs
smell. can compounds
Volatile originate from arecontamination in the field and
a group of carbon-based during
chemicals
storage
with low [90–92]. Volatile
molecular compounds
weight and highcan include
vapor alcohols,
pressure aldehydes,
produced hydrocarbons,
by bacteria and fungi acids,
as a
ethers, esters, ketones, terpenes, furans, sulfur, and nitrogen-containing
result of their metabolism, and numerous of VOCs can originate from contamination compounds. Fungi canin
produce
the fieldsimilar VOCs,storage
and during but the [90–92].
numbersVolatile
and the compounds
amounts of individual
can include VOCs vary. Differences
alcohols, aldehydes,
found in the global
hydrocarbons, pattern
acids, of VOCs
ethers, esters,are strictly correlated
ketones, terpenes, with
furans,fungi species
sulfur, andand strains and
nitrogen-con-
growth conditions, such as substrate, nutrients, pH, humidity, and temperature. An on-line
taining compounds. Fungi can produce similar VOCs, but the numbers and the amounts
VOC database (http://bioinformatics.charite.de/mvoc/index.php?site=home) (accessed on
of individual VOCs vary. Differences found in the global pattern of VOCs are strictly cor-
4 February 2023) reports more than 1000 VOCs from microorganisms; more than 300 of them
related with fungi species and strains and growth conditions, such as substrate, nutrients,
are classified as fungal VOCs [91].
Several VOC markers differentiating grains were identified [93,94]. Volatile organic
compound profile can be used as a fingerprint of different fungal species and toxigenic
or non-toxigenic strains [93,95–104]. The main VOCs found in cultures of fungi grown
on cereals belong to different categories, such as alcohols, aldehydes and ketones, ben-
Toxins 2023, 15, 146 6 of 17

zene derivatives, hydrocarbons, and terpenes [66]. Magan and Evans (2000) conducted a
milestone review of key studies carried out on the use of VOCs as potential indicators of
fungal activity, giving evidence of relationships between the metabolic pathway leading to
the formation of various VOCs and mycotoxin formation [66]. Since this review, a great
number of new studies have been carried out to identify fungal VOCs in various cereal
grains—grown under natural conditions or naturally infected. The most recent findings on
VOCs in fungi contaminated grains are reported in Table 1.

Table 1. VOC profiling due to fungal contamination in cereals: most recent acquisitions (not exhaus-
tive list).

Fungal
Samples Contamination VOC Analysis VOCs References
(*/**)
A total of 55 VOCs were identified.
Ethyl acetate-D and
3-hydroxybutan-2-one-D are
potential biomarkers specific to
Aspergillus flavus A. flavus contamination.
Maize GC-IMS [105]
(*) Aflatoxin B1 is positively
correlated with the level of
(E)-2-octenal-M, benzene
acetaldehyde, (E)-hept-2-enal-M, 2-
heptanone-D, and 2-pentyl furan.
A total of 11 VOCs were identified.
Octane,
2,2,4,6,6-pentamethylheptane,
decane, dodecane, toluene,
Jasmine brown Aspergillus oryzae
SPME/GC-MSD ethanol, 1-pentanol, 1-hexanol, [106]
rice (*)
1-octen-3-ol, 2-heptanone, and
2-pentylfuran could be used as
volatile markers for A. oryzae
contamination.
A total of 25 VOCs were identified.
Decanal, 1-octanol, 1-tridecanol,
nonanal, diethyl phthalate,
Aspergillus strains (A. candidus,
α-cedrene, cyclododecene, and
Rice A. fumigatus, and A. clavatus) HS-GC-MSD [107]
cis-thujopsene can be considered
(*)
as markers of infected rice
samples, with changes during the
storage period.
A total of 57 VOCs were identified.
Cyclooctasiloxane and
hexadecamethyl combination and
Ten fungal species, Alternaria (4),
pentadecane can be considered as
Cladosporium (3), Penicillium (2),
markers of early detection of
Wheat Aureobasidium (1), and GC-FID, GC-MSD [100]
postharvest fungi in grain for
Fusarium graminearum (1)
A. alternata and A. infectori,
(*)
respectively.
Naphthalene was identified only
in the headspace of C. herbarum
Toxins 2023, 15, 146 7 of 17

Table 1. Cont.

Fungal
Samples Contamination VOC Analysis VOCs References
(*/**)
A total of 23 VOCs were identified
(12 from dwarf and 15 from
hybrid maize).
Both varieties shared six common
markers: (+)-longifolene,
Fusarium graminearum and
Hybrid and β-farnesene, β-macrocarpene, and
F. verticillioides SPME/GC-MSD [108]
dwarf maize trichodiene.
(*)
Qualitative variability in VOCs
was observed upon infection of
different Fusarium species:
trichodiene was detected only
from F. graminearum.
A total of 46 VOCs.
Barley (malting Fusarium poae Volatile aldehyde fractions were
SPME/GC-MSD [109]
procedure) (*) influenced by F. poae
contamination during malting.
A total of 22 VOCs were identified.
3-hexen-1-ol, heptan-2-ol,
Fusarium graminearum, 1-octen-3-ol, octan-3-one,
SPME/GC-MSD
Maize F. verticillioides, and octan-3-ol, β-selinene, α-selinene, [110]
OLS/GC-MSD
F. subglutinans β-macrocarpene, and
β-bisabolene: markers for the early
detection of Fusarium infection.
A total of 29 VOCs were identified.
Levels of ethyl acetate, ethanol,
3-methylbutanol ethyl decanoate,
ethyl decenoate, 2-phenylethyl
SHS-SPME/GC-
Durum wheat Fusarium poae (*) acetate, 3-methylbutanal, hexanal, [111]
MSD
phenylethyl alcohol,
3-hydroxy-2-butanone, and acetic
acid changed as a function of time
after inoculation.
A total of 70 VOCs were identified.
Trichodiene, longifolene, 3-methyl
(**) butanal, tridecane, g-caprolactone,
DON < 1000 mg/kg; and 6,10,14-trimethyl-2-
Durum wheat 1000 mg/kg ≤ DON HS-SPME/GC-MSD pentadecanone: positively [112]
≤ 2500 mg/kg; associated with DON; Hexadecane,
DON > 2500 mg/kg. 2,3,7-trimethyl-decane, and
4,6-dimethyl-dodecane:
negatively associated with DON
A total of 46 VOCs were identified.
The most significant VOCs to
differentiate infected from
Barley, (**) analysis for trichothecenes A non-infected cereals: [E, E]-3,5
GC/MSD [100]
Oats, and rye and B octadien 2-one, 1-heptanol,
naphthalene, p-xylene and
dimethyl sulphone, and
trichodiene.
Toxins 2023, 15, 146 8 of 17

Table 1. Cont.

Fungal
Samples Contamination VOC Analysis VOCs References
(*/**)
Fusarium graminearum, A total of 16 VOCs were identified.
F. culmorum, F. cerealis, and 2-methyl-1-propanol,
Soft wheat SPME/GC-MSD [113]
F. redolens 3-methylbutanol, 1-octen-3-ol, and
(*) 3-octanone were infection-specific.
*: artificially inoculated; **: naturally contaminated; GC-IMS: Gas Chromatography–Ion Mobility Spectrometry;
SPME/GC-MSD: Solid-phase Microextraction/Gas Chromatography–Mass Spectrometry; HS-GC-MSD: Headspace-
Gas Chromatography–Mass Spectrometry; GC-FID: Gas Chromatography–Flame-ionization detection; GC-MSD: Gas
Chromatography–Mass Spectrometry; OLS/GC-MSD: Open-loop stripping/Gas Chromatography–Mass Spectrome-
try; SHS-SPME/GC-MSD: Static headspace–Solid-phase Microextraction/Gas Chromatography–Mass Spectrometry;
HS-SPME/GC-MSD: Headspace–Solid-phase Microextraction/Gas Chromatography–Mass Spectrometry.

Overall results indicate that (1) there is a wide range of fungal VOCs produced by
spoilage fungi; (2) VOCs can be used as taxonomic markers of fungal species; (3) the pres-
ence of VOCs in naturally contaminated grain can be used as an early indicator of spoilage;
and (4) more than single VOCs, the analysis of the VOC profile, by using multivariate
analysis techniques, represents a powerful tool for the early detection and time evolution
of fungal spoilage.
Gas chromatography (GC)-based techniques have been used for the specific and sensi-
tive analysis of VOCs and the volatilome profile. These techniques are reliable, specific, and
sensitive, but expensive, time-consuming, and labor-intensive. In this scenario, according
to the need of the feed industry, e-nose may represent a powerful tool for a rapid and on-site
analysis of VOC profiles for identification of fungi contamination in agricultural commodi-
ties. Rapid analysis of mouldy and mycotoxin-contaminated agricultural commodities can
reduce the risk of human/animal exposure to mycotoxins. Electronic nose was success-
fully used for VOC analysis and the early detection and differentiation between spoilage
fungi and mycological quality grading of barley grains [65,114]. The study of Keshri and
Magan [67] was the first one that showed that e-nose was able to differentiate between
mycotoxigenic and non-mycotoxigenic strains of Fusarium moniliforme and F. proliferatum
on the basis of their VOC production patterns [115]. From these studies, research on this
topic has developed and increased. E-nose showed a very good discrimination capability
for grain quality discrimination and detection of fungal contamination of cereal grain by
discriminating contaminated and non-contaminated grains by Penicillium and Fusarium
spp. and changes during the crucial stages of fungal growth [65–123].
By using an e-nose, the volatile compounds released by four Fusarium species were
studied, and infected and non-infected wheat grains in the post-harvest chain were differ-
entiated [113]. E-nose, combined with GC-MS, was able to identify the changes of volatile
profile due to Aspergillus spp. growth in rice kernels [122]. Visualization of VOCs profiles
of Aspergillus oryzae contaminated brown rice was possible and useful for early detection of
fungal infection [106]. A systematic review on detection and identification of fungal species
by e-nose technology in various fields of application beyond that of food safety has been
recently published [83].
In addition to research on fungal VOCs as indirect indicators of fungal growth, in
recent years, studies on fungal VOC production explored new topics: role in ecosystems
(many ecological interactions among fungi and plants, arthropods, bacteria, and other fungi
are mediated by VOCs), development of environmentally friendly biopesticides, and use
in biotechnological applications (biofuel, biocontrol, and mycofumigation) [10,123–125].
Moreover, there is increasing experimental evidence that some fungal VOCs may be toxic.
Bennett and Inamdar (2015) proposed the term “volatoxin” to describe VOCs with toxigenic
properties [89].
Toxins 2023, 15, 146 9 of 17

6. E-Nose for Mycotoxin Detection

Rapid evaluation of feed quality and safety represents a challenge for the feed industry
for mycotoxin risk management. As previously discussed, the potential for using sensor
arrays to discriminate between toxigenic and non-toxigenic fungi exists [96]. Detection
of mycotoxin contamination by e-nose is based on the detection of changes in the compo-
sition of VOCs produced by mycotoxigenic fungi during their growth and biochemical
processes. Terpene production has been found to correlated to the production of AFB1 [104].
The volatile terpene trichodiene is the first metabolite in the trichothecene biosynthesis
pathway [126]. The production of volatile terpenes relates to the formation of Fusarium
trichothecene mycotoxins [127–129]. Volatile sesquiterpene hydrocarbon has been found
to be a marker for Penicillium roqueforti strains, producing PR toxin [130]. No volatile
compound uniquely related to OTA formation has been found [131]. The content of DON
in durum wheat has been found to be positively (trichodiene, longifolene, 3-methyl bu-
tanal, tridecane, g-caprolactone, and 6,10,14-trimethyl-2-pentadecanone) and negatively
(hexadecane, 2,3,7-trimethyl-decane, and 4,6-dimethyl-dodecane) correlated to the pattern
of VOCs [112].
Recent applications reporting specific applications of e-nose for mycotoxin detection
are reported in Table 2.

Table 2. Application of e-nose for mycotoxin detection in cereals.

Sample E-Nose/Sensor
Mycotoxins Data Analysis Tested Hypothesis References
(*/**) Array
Fox 3000/(6 SnO2
Aflatoxins—two classes:
and 6 CTO);
AFs Maize (*) SVM, k-NN below and above [132]
Cyranose 320; and
10 µg/kg (ppb)
DiagNose/12 MOS
Discrimination among four
AIR PEN 3/10 DON contamination
DON Wheat (**) CART [133]
MOS thresholds: 1750, 1250, 750,
and 500 µg/kg
AIR PEN 3/10 Discrimination at levels above
AFB1, FUM Maize (**) ANN, LR¸ DA [134]
MOS or below the legal EU limits #
Three classes of
contamination: below the EU
AIR PEN3/10
Afs, FBs Maize (**) DFA regulatory limits ##, [135]
MOS
single-contaminated, and
co-contaminated
Two contamination classes:
AOS-ISE Nose
DON Wheat bran (**) DFA A: DON ≤ 400 µg/kg and B: [136]
2000/12 MOS
DON > 400 µg/kg
Three contamination classes:
AOS- ISE Nose A: DON ≤ 1000 mg/kg; B:
DON Durum wheat (**) DFA [112]
2000/12 MOS 1000 < DON ≤ 2500 mg/kg;
and C: DON > 2500 mg/kg.
Three clusters based on the
DON content proposed by the
European legislation: A:
AIR-PEN2/10 non-contaminated; B:
DON Durum wheat (**) PCA, CART [137]
MOS contaminated below the limit
(DON ≤ 1750 µg/kg); and C:
contaminated above the limit
(DON > 1750 µg/kg)
Toxins 2023, 15, 146 10 of 17

Table 2. Cont.

Sample E-Nose/Sensor
Mycotoxins Data Analysis Tested Hypothesis References
(*/**) Array
FBs: low content below
1.6 mg/kg (average
FBs Maize (*) EOS835 /6 MOX PCA, PLS [138]
1.0 mg/kg) vs. high content
above 1000 mg/kg
AIRSENSE Aflatoxin-containing samples
AFs Maize (**) PCA, LDA [139]
PEN2/10 MOS and aflatoxin-free samples
FOX 3000,
OTA, citrinin time changing
OTA, citrinin Durum wheat (**) Alpha-MOS/12 CORR [140]
during storage (25 weeks)
sensors
DON-containing samples and
DON Durum wheat (**) PEN2/10 MOS PCA, MR [141]
DON-free samples
The OTA level varied between
VCM 422/10
0 and 934 mg/kg; the DON
DON, OTA Barley (**) MOSFET, 6 SnO2 , PCA, PLS [142]
content varied between 0 and
and 1 Gascard CO2
857 mg/kg
AFs: aflatoxins; DON: deoxynivalenol; FBs: fumonisins; OTA: ochratoxin; DFA: discriminant function analysis;
PCA: principal component analysis; CART: classification and regression tree analysis; PLS: partial least squares
analysis; LDA: linear discriminant analysis; CORR: correlation analysis; SIMCA: soft independent modelling of
class analogy; * artificially inoculated; ** naturally contaminated; SnO2 : oxide sensors; CTO: chromium titanium
oxide sensors; MOS: metal–oxide sensors; MOSFET: metal–oxide semi-conductor field-effect transistor sensors;
SVM: support vector machine; k-NN: k-nearest neighbour; ANN: artificial neural network; LR: logistic regression;
DA: discriminant analysis; DFA: discriminant function analysis; MR: multiple regression analysis; # AFB1 :5 µg/kg,
FBs: 4000 µg/kg; and ##: AFs < 5 ppb, FBs FM < 4 ppm.

E-nose can be a powerful tool for quantitative/semiquantitative prediction of my-

cotoxin levels in grains. To move e-nose analysis from research to the industrial level,
there are several questions that need answers. The main points that must be considered
are: (1) the presence of maximum levels for mycotoxins in feed for practical enabling of
rapid decision-making regarding the acceptance or rejection of lots of cereal and ensuring
safety standards; (2) mycotoxin co-contamination; and (3) the classification and prediction
accuracy of the e-nose-based model.
Regarding the first point, e-nose was able to predict the FB content of maize cul-
tures for high and low contamination levels [138]. E-nose was able to discriminate
DON-contaminated and non-contaminated wheat and aflatoxin-contaminated and non-
contaminated maize [139,141]. E-nose analysis was able to discriminate durum wheat
samples at contamination levels close to that of the DON maximum limit set by the EU
regulations [111,136]. E-nose was able to detect OTA and to predict whether the OTA level
was below or above 5 ug/kg, representing the maximum level for OTA in cereals for food
by EU regulations [140].
Mycotoxin co-contamination is the rule. E-nose analysis has been proposed to de-
tect aflatoxin and fumonisin co-contamination in maize [134]. E-nose was effective in
detecting co-contaminated samples, but with a low classification accuracy of 61% and 67%,
respectively, of samples correctly classified for co-contamination using LDA.
The type and percentage of misclassified samples are important and are critical issues
in determining the performance and accuracy of e-nose analysis. Olsson et al. (2002)
investigated the possibility of using fungal VOCs as indicators of two mycotoxins (OTA
and DON) in barley, using both e-nose and GC/MSD [140]. In that study, the authors
reported that the e-nose misclassified less than 20% of samples in the case of OTA. The
DON level could be estimated using a partial least square (PLS) model constructed using
the sensor signals from the e-nose. The detection of co-contamination was not the aim of
this study, although several samples were found to be contaminated by both OTA and
DON. Overall results indicate that, for single mycotoxin contamination, high discrimination
Toxins 2023, 15, 146 11 of 17

accuracy between contaminated and non-contaminated grain has been reported. However,
when mycotoxin co-contamination occurs, the predictive accuracy of e-nose is still limited
and unsuitable for industrial applications in a real context.
Finally, several e-noses are available on the market, and they can be customised accord-
ing to the need. The very recent study of Machungo et al. (2022) compared the performance
of three e-nose instruments for the detection of VOCs in maize contaminated with aflatoxins
(Table 2) [132]. One of the three tested instruments (DiagNose) was more effective than the
other two (Fox 3000 and Cyranose) for the detection of aflatoxin contamination of maize,
with a cross-validated classification accuracy for the different sample classes ranging from
81% to 94%.

7. Conclusions
E-nose represents a powerful tool in the feed chain for quality and safety control
and monitoring. E-nose offers potential as a rapid and cost-effective diagnostic tool for
mycotoxin contamination screening at the market entry level. However, before e-nose
laboratory-based assays can move from research into the feed industry and become a reality,
we must face and overcome several challenges to improve e-nose performance.
The future challenges are: the sensor materials, data analysis, pattern recognition
systems, and a better understanding of the industrial needs related to safety and quality
control of the feed supply chain. A universal e-nose for mycotoxin detection is not realistic;
a unique e-nose must be designed for each specific application. Limitations still exist
regarding sensitivity and selectivity of sensors. The major drawback is represented by
sensors’ sensitivity to environmental conditions, particularly humidity and temperature.
Improved modelling, correlation between chemical markers and sensor responses, and
robust and suitable e-nose methods and advancements in signal processing algorithms
must be validated for specific needs. In the field of mycotoxin co-contamination detection,
the predictive accuracy of the e-nose models is still limited for industrial applications in
a real context. Last, but not least, specific sampling models must be carefully selected to
enhance the accuracy of e-nose analysis.
Supplementary Information could be found in Figure S1.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/toxins15020146/s1, Figure S1. Chemical structures of the mycotoxins.
Author Contributions: Conceptualization, F.C.; methodology, F.C., M.O., F.C., M.O., F.F. and S.M.;
writing—original draft preparation, F.C., M.O., F.F., S.M. and L.F.; writing—review and editing, F.C.,
M.O. and L.P.; supervision, F.C.; funding acquisition, F.C. and L.P. All authors have read and agreed
to the published version of the manuscript.
Funding: “One Health Action Hub: University Task Force for the resilience of territorial ecosystems,”
funded by the Università degli Studi di Milano (PSR 2021-GSA-Linea 6).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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