Trends in Food Science & Technology xxx (xxxx) xxx

Contents lists available at ScienceDirect

Trends in Food Science & Technology

journal homepage: www.elsevier.com/locate/tifs

Uncovering the dormant food hazards, a review of foodborne microbial

spores’ detection and inactivation methods with emphasis on their
application in the food industry
Mohamed A. Farag a, b, *, Matta A. Mesak b, Doaa B. Saied b, Nada M. Ezzelarab c
a
Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, 11562, Egypt
b
Chemistry Department, School of Sciences & Engineering, The American University in Cairo, New Cairo, 11835, Egypt
c
Biology Department, School of Sciences & Engineering, The American University in Cairo, New Cairo, 11835, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Spores are dormant structures that are widespread in various foodstuffs leading to food spoilage or
Bacterial spores foodborne illness. Many attempts have been made to eliminate and minimize spores in food with though inac­
Fungal spores tivation processes not always efficient. To overcome the limitations of inactivation procedures, detection
Inactivation
methods for spores are warranted to avoid its hazards. Scope and approach: In this study, we present a
Detection
Food hazards
comprehensive state of the art review on the various inactivation procedures i.e., radiation, heat, chemical, and
Food industry sporicidal agents or combined technologies with a focus on their advantages and limitations. Finally, a review of
the different detection methods including molecular, microscopic, spectroscopic, biosensors, and immunoassays
is presented highlighting their applications in the food industry.
Conclusions: In conclusion, providing such state-of-the-art comparison of the various technologies for spore
detection and inactivation shall aid food microbiologists and quality control specialists to choose the most suited
one for spore’s prevention in different food matrices. Combining two or more detection techniques appears to
enhance the sensitivity or to overcome some drawbacks and limitations within each method.

1. Introduction adverse environmental stresses such as heat and chemical treatment

Spores are dormant, tough, and non-reproductive structures pro­
duced by both fungi and certain bacteria (Benedict, Chiller, & Mody,
2016; Kort et al., 2005), to represent one of the most common hazards in
foodstuffs that can lead to food spoilage or food-borne illnesses (Kort
et al., 2005). Bacterial spores can be produced by different types of
bacteria including anaerobic (Clostridium botulinum), aerobic (Ther­
moactinomyces vulgaris), or facultative aerobic bacteria (Bacillus cereus)
(Soni, Oey, Silcock, & Bremer, 2016). Bacterial spores can be found in
different food types including meat, vegetables, dairy products, grains,
etc. (Eckert, Burghoffer, & Barbut, 2013; Kaneko et al., 2011;
López-Enríquez, Rodríguez-Lázaro, & Hernández, 2007). In contrast,
fungal spores can be produced by all fungi types such as Aspergillus flavus
and they are likely to produce mycotoxins such as aflatoxins (Bansal,
Mangal, Tushir, Oberoi, & Gupta, 2019). Compared to fungal spores,
bacterial spores are more durable to the adverse environmental changes
(Huang & Hull, 2017). Spores are known to be highly resistant to
(Kort et al., 2005). (see Fig. 1)
There is a 7 steps-cycle that describes the changes of vegetative cells
to form spores. The starting step to complete the cycle forming a spore is
a normal vegetative cell represented as 0 stage, followed by stage I/II,
where asymmetric cell division occurs with two partitions, the smaller
compartment is termed as prespore. Passing through the following step,
stage III, the prespore form a distinct cell called forespore by the
engulfment of the mother cell. Syntheses of the spore cortex occur in
stage IV, and after which the coat of the spore is formed in the pro­
ceeding step, stage V. When stages IV and V take place, production of
specific compounds such as Dipicolinic acid (DPA) occurs. The spore
becomes further mature during stage VI with condensed molecules that
are prominent. The release of matured spore follows the mother cell
degradation in the final stage VII. The final step represents the protective
shield of its internal dormant microorganism content till conditions are
favored again for the growth of its vegetative form. (Leggett, McDonnell,
Denyer, Setlow, & Maillard, 2012; McDonnell, 2020).

* Corresponding author. Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, 11562, Egypt.
E-mail address: mohamed.farag@pharma.cu.edu.eg (M.A. Farag).

https://doi.org/10.1016/j.tifs.2020.10.037
Received 8 August 2020; Received in revised form 18 October 2020; Accepted 24 October 2020
Available online 6 November 2020
0924-2244/© 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Mohamed A. Farag, Trends in Food Science & Technology, https://doi.org/10.1016/j.tifs.2020.10.037
M.A. Farag et al.
Bacterial spore structure differs from that of the vegetative cell and
that is how it gains its resistance by limited permeability and less water
content. The innermost layer is the core where DNA, RNA, ribosomes,
some enzymes are present with a high content of the DPA mostly in
chelated form. The cortex and germ cell wall come after that, composed
of peptidoglycans followed by the coat, which is described as a lamellar
layer, mainly protein structured with minor carbohydrate content. Its
structure differs between bacterial species and strains of the same spe­
cies as well. Exosporium is the outermost layer, of which some bacterial
spores lack this layer. It is also composed mainly of protein with lipids
and carbohydrates in lesser amounts (Leggett et al., 2012).
In contrast, fungal spores, the reproductive route for fungi, are pro­
duced either sexually or asexually and they can be unicellular or
Fig. 1. Sporulation stages, represented with symbols indicating each stage.
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Trends in Food Science & Technology xxx (xxxx) xxx
multicellular depending on the fungus type (Sandle, 2019). Morpho­
logically, fungal spores show a wide range of shapes, sizes, and external
structures that affect their texture as well (Crandall, Saarman, & Gilbert,
2020). Mostly, the spores’ outer layer is composed of hydrophobic
proteins and melanin in the shape of rodlets. Such a layer acts as a
permeability barrier to prevent germination (El-Enshasy, 2007). How­
ever, due to the vast variations among fungal species (≈100 thousand
accepted species (Hyde et al., 2020)), there is not a general cellular
structure mentioned in the literature describing the fungal spore.
According to the Center for Disease Control and Prevention, United
States reports ca. 128,000 cases of food-borne diseases with about 3000
deaths occur yearly (“Burden of Foodborne Illness: Findings,”). The
mortality, morbidity and economic burden attributed to foodborne
M.A. Farag et al.
diseases are posing an increasing threat to the food industry and health
sectors (Havelaar et al., 2015).
Food spoilage represents another burden due to food loss with
increasing limited sources of nutrition worldwide (Rawat, 2015). One of
the major causative agents of food spoilage is derived from bacterial
infection (Kort et al., 2005), warranting for developing methods to
detect foodborne pathogens at earlier stages in food production (Soni
et al., 2016). As a result of the huge impact of spores on health and
economy, methods for detecting spores at their earlier stages with
minimal preparation, and without its germination is faced by several
challenges. This review focuses on the different detection methods for
spores in food highlighting its application type, advantages, and or
limitations. Finally, inactivation methods for spores are discussed
highlighting its application in different food types.
This review presents the first comprehensive state of the art compile
on the various spores’ inactivation procedures along with the different
detection methods i.e., molecular, microscopic, spectroscopic, bio­
sensors, and immunoassays is presented highlighting their applications
in the food industry. Search was conducted from two electronic search
engines including PubMed and Web of Science. The following search
terms were used (“Bacterial spores” AND “fungal spores” AND “inacti­
vation” AND “detection” AND “food hazards”.
The search yielded 419 papers and a total of 314 papers were
excluded according to inclusion and exclusion criteria with around 105
papers cited in this review. We screened the titles and abstracts of the
search results according to our inclusion/exclusion criteria. Original
articles stating information about inactivation and detection methods of
spores in food were included. Exclusion criteria included overlapped
data sets, articles with only abstracts, thesis, reviews, conference papers,
case reports, and articles in languages other than English.
A list of chemical and physical methods for inactivating bacterial and
fungal spores in different food matrices is presented in Table 1 & Fig. 3.
1.1. Thermal
Achieving a targeted spore inactivation level by applying thermal
technique is considered crucial to assure safety sufficient log reduction
of spores. For example, in a rice sample (Fig. 3), cooking inactivated
B. cereus spores achieved around 4 logs of reduction within 256, 138,
and 44 min at 90.5 ◦ C, 95 ◦ C and 99 ◦ C, respectively. Although the
importance of thermal-death-times in constructing food lethality re­
gimes in HACCP (Hazard Analysis Critical Control Point) plans,
increased heating level and contact time could affect food quality such
as color, taste, texture, and aroma (Juneja, Osoria, Hwang, Mishra, &
Taylor, 2020).
Another method for implementing heat in food i.e., cocoa powder is
within the roasting step as part of its processing. The needed time to
achieve the first log reduction, δ, was calculated for forms of cocoa,
beans, and nibs, at three different temperatures with two inoculated
spore-forming bacteria. In case of B. cereus addition to cocoa beans, the δ
value at 110 ◦ C, 125 ◦ C, and 140 ◦ C was around 149, 56, and 23 min,
while the values at the same set of temperature for crushed bean, the nib
was at 92, 31, and 17 min, respectively (Table 1). In case of Geobacillus
stearothermophilus spores, one of the common highly heat resistant
spore-forming bacteria found in food manufacturing facilities and
products, log reduction needed time values at same set of temperature
was around 105, 40, and 30 min for nibs versus 184, 76, 49 min for
beans, respectively. Moreover, roasting not only has decreased cocoa
pH, but also water activity nearly to the half. Reduction of water activity
helps prevent microorganisms contamination in foodstuff; however, it
negatively affects heat transfer increasing bacterial spore tolerance
(Pereira, Stelari, Carlin, & Sant’Ana, 2019; Syamaladevi et al., 2016).
Increasing roasting time at high temperature may not support a suffi­
cient reduction of spore’s bioburden. Further inactivation steps using
other techniques should be thus considered regarding the remaining un
inactivated spore count (Pereira et al., 2019), and suggestive that
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Trends in Food Science & Technology xxx (xxxx) xxx
implication of more than one approach may be more efficient in spores
inactivation as discussed later.
Pasteurization is a commonly used method in the dairy industry, in
which exposure time to high temperature is rather short. Dairy products
manufacture, particularly milk apply sequential heating after pasteuri­
zation at 70 ◦ C followed by heat treatment at 125 ◦ C for 15 s as a
microbiological control regime. With pasteurization, some thermoduric
spores forming bacteria which can tolerate high temperature though can
thrive this process. The strategy of using sequential heating asides from
its effect on decreasing microbial vegetative count is to germinate spores
which later by further treatment to be inactivated and to ultimately lead
to a reduction in spores count. Exposure to extreme heat treatment such
as 125 ◦ C for 15 s would result in a change in milk protein structure
affecting its quality asides from several physical attributes and chemical
changes i.e., Maillard reaction affecting milk acceptability (Sanchez
Alan, Wang, & Schmidt, 2017; Van Boekel, 1998). Such limitation in
applying temperature especially in case of food containing thermolabile
dietary chemicals warrant for developing or to modify spore inactivation
methods (F. Li et al., 2020).
1.2. Thermal combined methods (hurdle technology)
To tackle the limitation of applying high thermal treatment,
combining other physical parameters, such as pressure, is considered
what is termed as hurdle technology (Mukhopadhyay & Gorris, 2014).
Hurdle technology is a method of ensuring the safety of foods by elim­
inating or controlling the growth of pathogens, making the food safe for
consumption, and extending its shelf life through the application of a
combination of technologies and approaches Applying high pressure at
300 and 600 MPa (Table 1) in parallel with thermal heat at 70 ◦ C
inactivated Alicyclobacillus acidoterrestris spores which may exist in
apple or orange juices (Fig. 3) by around 2.5 and 2.4 log reductions
within 5 min. Moreover, if heat shock was applied prior to counting and
after heat and pressure application, spore count showed a reduction by
1.7 times than without heat shock revealing the high-pressure effect to
increase spores’ sensitivity for heat shock inactivation facilitating the
partial germination of spores. By converting the spore form of bacteria to
the sporulation stage, resistance to inactivation would decrease result­
ing in a false high inactivation rate that is not due to heat and pressure
exposure only, but rather for the further heat shock suggestive to apply
heat after applying pressure and heat together for further inactivation
(Ribeiro & Cristianini, 2020).
Applying 20 MPa pressure of CO2 gas plus high temperature at
around 85 ◦ C for 60 min and centrifugation led to spores’ inactivation.
However, a significant portion of the dead spores that maintained
chelating dipicolinic acid with Ca2+ as (CaDPA) germinated with
blockage of outgrowth. Dipicolinic acid is mainly located in the spore
core and converting it to CaDPA will facilitate its release and replace­
ment of the core instead with water inside spore’s core aids in its
germination phase 1. This combination minimized mutation, damaged
the inner membrane (IM) of B. cereus and prevented the outgrowth after
germination (Rao et al., 2019).
Further addition of ultrasonic source to thermal treatment and
pressure (mono thermosonication MTS) exhibited a synergistic spori­
cidal action on B. cereus spores (Table 1). Exosporium, an outer spore
layer detachment was promoted using ultrasonic with which spore
adhere effect to surfaces was lessened. MTS inactivated spores by 3.1 log
reduction with a high rate of spore damage. This treatment combination
also exhibited significant changes in the spore intracellular structure
where destruction for both coat and cortex occurred. Recently, several
trails of using non-thermal techniques as an alternative for thermal
treatment by different approaches such as heat intensity reduction are
employed. These methods include HPP accompanied by heat, high-
pressure CO2, and radiation. Incorporation of ultrasound reduced en­
ergy, consumption of water, and released greenhouse gases. these pose
the technique to be applied as a green and safe combination that may be
M.A. Farag et al. Trends in Food Science & Technology xxx (xxxx) xxx

Table 1
Chemical and physical methods for inactivating bacterial and fungal spores.
Inactivation Microorganism/strain Food type Results/log reduction Advantages Disadvantages References
method

Thermal B. cereus Rice 4 log reduction Design food lethality ↑Time of Juneja et al. (2020)
inactivation processes inactivation
Roasting process B. cereus Cocoa bean & δ → 92, 30, and 17 min in cocoa Limited effect Pereira et al. (2019)
G. stearothermophilus nib nib
δ → 148, 55, and 23 min in cocoa
bean
Sequential heating B. licheniformis Milk 1st heating → spore germination Applied for milk Protein damage (F. Li et al., 2020)
processes 2nd heating → inactivation products
(HPT) C. botulinum ↓Acidic HPT→ ↓ detection limit ↓ Holding times & Maier, Schweiger,
foods over processing Lenz, and Vogel
(2018)
(HPP) and (HPP + Alicyclobacillus N/A − 300 and 600 MPa/5 min failed (HPP + T) → Needs high temp Ribeiro and Cristianini
T) acidoterrestris to inactivate spores efficient spore (2020)
±heat shock -HPP at 70 ◦ C→2.5 log (300 MPa) inactivation
and 2.4 log (600 MPa)
(HPCD + HT) B. subtilis N/A -Most pelleted spores and all light -↓ spore’s strains Rao et al. (2019)
spores were killed mutants
- blocking outgrowth
(US) + thermal B. cereus N/A MS → 92.5% DPA release Green sterilization Lv et al. (2019)
(TS), (MS), & MTS →3.12 log reductions technology
(MTS)
TS B. subtilis N/A -Synergistic inactivation Food industry Spore Fan et al. (2019)
TS→ 1.8 ± 0.14 log germination
US + heat B. subtilis Water milk rice US % D-value reduction→35, 18, Good inactivation Commercially Ansari et al. (2017)
4 (milk, water & rice) method not viable
(UVC) radiation B. subtilis Milk 1-6 log CFU/mL reduction in Low temp for Low Ansari et al. (2019)
skim, whole bovine, and ovine inactivation transmittance
milk
Pressure & heat B. coagulans Acidic food 1.5–3.4 logs reduction, Effective Daryaei and
<10 CFU/mL Balasubramaniam
No growth after storage (2013)

SAEW + BAC and B. cereus N/A SAEW + 60 ◦ C → 0.76& 0.59 log Effective cleaning Hussain et al. (2019)
mild heat CFU/ml. against B. cereus in
SAEW + BAC +60 ◦ C → 1.9 &1.9 food contact
log CFU/ml surfaces
(HPCD + HT) B. subtilis N/A - HPCD + HT→ efficient Protective effect Rao et al. (2015)
inactivation on the spores
- HPCD + HT→90% of (DPA)
(MS) and (MTS) B. mycoides, Pasteurized 4 log reduction→ Spore thermal Condón-Abanto,
B. weihenstephanensis, and crab meat B. weihenstephanensis resistance Arroyo, Álvarez,
P. psychrodurans MTS→↑% synergism of Condón, and Lyng
inactivation (2016)
Temp + pressure at B. subtilis N/A (2.4–4.9) log at 150 ◦ C, 120 s and ↑aw level→↑spore Hauck-Tiburski,
different aw aw 0.5 inactivation. Rosenthal, Iaconnelli,
Perrier-Cornet, and
Gervais (2019)
(DM) (RO; DM), B. subtilis Whey lactose DM→↑influence ↓aw. →↑ whey Marx and Kulozik
(NF; DM) and RO & NF/↑ DM contents →↑ protein & lactose (2018)
(UF; DM) spores’ stability level
(HPP) and power Clostridium perfringens, B. Fruit, beef, C. Perfringens: HPTP, TS, -Inactivation of Evelyn, Milani, & Silva
ultrasound + cereus strawberry thermal→2, 1,0 log bacteria, mold, and (2017)
heat (HPP- Alicyclobacillus puree, B. cereus: HPTP, thermal→4, 0.3 yeast.
thermal or HPTP, acidoterrestris, milk, log -↓Inactivation time
TS) Byssochlamys nivea orange juice N. fischeri: HPTP, thermal→4.3, & ↑ food quality
Neosartorya fischeri 0 log
Saccharomyces cerevisisae S. cerevisiae: HPP, TS, thermal→3
log
(HPP) combined B. cereus ↓ acidic food - HPP (200–600) MPa → ↓ spore HPP ↑ thermal ↑Specific energy Evelyn and Silva
with a thermal reconstituted numbers slightly inactivation than the thermal (2015)
process milk - 600 MPa HPP + heat → processing
improved spore inactivation in
milk
(TC) and (HP) B. cereus Reconstituted HP& TC→ 2.4 and 3.1 log -Synergistic TC→ cinnamon- Cetin-Karaca and
infant formula TC + HP→2 log antimicrobial effect like taste Morgan (2018)
(UV) + gaseous I2 Btk & N/A -Btk spores with UV and iodine on -Effective The filter type Nakpan et al. (2019)
B. antracis A. fumigatus MCE filter produced a synergistic -Used in air handling was a factor
effect filters of production
areas
(UV-LEDs) A. niger, P. polonicum Water - UV/LEDs showed stronger -Alternative for The electrical Wan et al. (2020)
T. harzianum inactivation to 254 nm (LP) water disinfection consumption of
- Efficient UV-LEDs was
high
(continued on next page)

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Table 1 (continued )
Inactivation Microorganism/strain Food type Results/log reduction Advantages Disadvantages References
method

(UV) and (UV- B. cereus species Drinking water UV dosage 180 mJ/cm2 → (3 log) Less UV dose Radiation Zeng et al. (2020)
AOPs) (UV/ reduction. prevented spore’s exposure
H2O2 and UV/ regrowth
PMS)
(UV/PMS) Trichoderma sp., Water (UV/PMS) >UV in inactivation Control fungi in Resistance of Wen, Xu, Zhu, et al.
Acremonium sp., Penicillium rate constant drinking water fungi to UV (2017)
sp., Cladosporium sp
(UV)-based (AOP) B. subtilis Water UV265/Cl2, UV280/Cl2 →1.8, 1.5 UV/Cl2→ Synergism (G.-Q. Li et al., 2018)
log
RF heating (B. cereus) Red pepper 90 ◦ C → 4 log reduction (aw: 0.70) Control spore- Jiao et al. (2019)
powder forming pathogens
in (LMFs)
(MCPT) B. cereus Pepper flakes -MCPT →2.6 log with ↑ aw Same color of red Kim et al. (2017)
-Vacuum-dried red pepper→↑ pepper
reduction
Microwave C. difficile N/A Complete inhibition of spore Simple and time- Ojha et al. (2016)
irradiation viability at 107 CFU/ml at 800 W efficient
(UV–C) & (US) Alicyclobacillus Fruit juices US→↓ inactivation impact >95 ◦ C thermal No synergism Tremarin, Brandão,
acidoterrestris UV-C→↓ spores inactivation and Silva (2017)
(UV–C LEDs) Penicillium expansum Apple UV–C LEDs→ > 2 log CFU No change in (Rios de Souza,
physicochemical Popović, Warriner, &
properties Koutchma, 2020)
(PL) B. subtilis & G. N/A Spores of G. stearothermophilus Vegetative cells and ↓sensitivity (Artíguez & Martínez
stearothermophilus were ↓ resistant to PL spore’s inactivation de Marañón, 2015)
(IPL) B. subtilis N/A IPL (7.40 J/cm2) →7 log ↑ log reduction Resistance of Jo et al. (2019)
reduction spores
PEF B. cereus Milk -High resistance of spores to PEF Synergistic effect Spore resistance Bermudez-Aguirre
alone with nisin et al. (2012)
Surface of small B. subtilis N/A 10 ​ mm ​ gap ​ distance Humidification→ Tanino, Matsui,
LPDBD̅̅̅̅̅̅̅̅̅̅̅̅̅̅→3.6 ×
spheres by (DBD) ↑effectiveness Uehara, and Ohshima
10− 4 survival ratio
6 ​ mm ​ gap ​ distance
(2020)
LPDBD̅̅̅̅̅̅̅̅̅̅̅̅̅̅→ no
viable cells
Photo electro P. expansum N/A Photo electro catalytic process → Spores in cold Su et al. (2020)
catalytic process 1 log reduction environment
(ASPEC) cell
Photodynamic A. flavus Maize kernels 3 & 2 log CFU/ml in suspension Effective spore Temba et al. (2016)
inactivation and maize kernels inactivation method
(NTP) reactor + Aspergillus sp Black pepper -NTP → (3 Log) reduction No change in ↓ antioxidant Tanino et al. (2019)
humidified gases – piperine level properties
Cold plasma B. subtilis N/A N/A No change in Inactivation Mendes-Oliveira et al.
sterilization properties resistance (2019)
(PAW) B. cereus N/A PAW→1.6–2.9 log reduction Bai et al. (2020)
(PAA) or (H2O2) G. stearothermophilus N/A 0.06% PAA →5-log reduction Curves should be Hayrapetyan et al.
considered (2020)
Chlorine dioxide Cladosporium sp. Water ClO2 > Cl2 in inactivation efficacy ClO2 unstable Wen, Xu, Huang, Zhu,
Trichoderma sp. Penicillium at↑ conc. and Ma (2017)
sp.
Surfactant C. sporogenes N/A 1–2% surfactant components Effective Cho and Chung (2018)
were 1.5–2.5 log
Bacteriocin G. stearothermophilus Canned Enterocin AS-48→ ↓ viable cell Bio preservative (Pilar Martínez
enterocin AS-48 food detection level Viedma et al., 2009)
Enterocin EJ97 G. stearothermophilus Canned food Germinated endospores → Bio preservation (P. M. Viedma et al.,
bacteriocin sensitive 2010)
Chemical B. thuringiensis B. anthracis N/A Strong spore inactivation Raman compatible -Time dependent Stockel et al. (2010)
(Formaldehyde, method -Spore resistance
Danchlorix, PAA)
Chemical, Fogged Bacillus atrophaeus Bacillus N/A Fogging→ >6 LR Decontamination Incomplete Richter, Wood,
(PAA)/(H2O2) anthracis Ames % decontamination inactivation Wendling, and Rogers
efficacies→38%, 41% (2018)
Chemicals B. cereus, B. subtilis B. N/A 0.05% → > 3 Log10,91% of the Anti-Bacillus agent Alqadeeri, Rukayadi,
(β-asarone, and pumilus & B. megaterium spores Abbas, and Shaari
asaronaldehyde) (2019)

Abbreviations: (δ) time needed for the 1st log-reduction, (aw)water activity, all-solid-state photo electrochemical (ASPEC), An ultraviolet (UV)-based advanced
oxidation process (AOP), adenosine triphosphate (ATP), Bacillus thuringiensis var. kurstaki (Btk), Bacillus subtilis (B. subtilis), Bacillus cereus (B. cereus), Bacillus antracis
(B. antracis), B. amyloliquefaciens Bacillus. Amyloliquefaciens, benzalkonium chloride (BAC), Clean-in-Place (CIP), Clostridium difficile (C. difficile), Clostridium sporogenes
C. sporogenes, Clostridium botulinum C. botulinum, cold atmospheric pressure plasma (CAPP), deoxyribonucleic acid (DNA), dielectric barrier discharge (DBD), dry
matter (DM), dipicolinic acid (DPA), Geobacillus stearothermophilus G. stearothermophilus, green fluorescent protein (GFP), high pressure (HP), high pressure CO2
combined with high temperature (HPCD + HT), high pressure thermal (HPT), high pressure processing (HPP) and high pressure processing combined with temperature
(HPP + T), high pressure CO2 (HPCD) at high temperature (HT) (HPCD + HT), hydrogen peroxide (H2O2), inner membrane (IM), Intense pulsed light (IPL), low-
moisture foods (LMFs), LPUV (low pressure ultraviolet), low-pressure mercury (LPM), Low-pressure DBD (LPDBD), low pressure double inductively coupled
plasma (DICP) system, log reduction (LR), A mixed cellulose ester (MCE), Microwave-combined cold plasma treatment (MCPT), (manosonication, MS), (man­
othermosonication, MTS), nonthermal plasma (NTP), nanofiltration (NF), peracetic acid (PAA), pulsed light (PL), pyridine-2,6-dicarboxylic acid (DPA), Plasma
activated water (PAW), pulsed electric fields (PEF), personal protective equipment (PPE), powdered infant formula (PIF), Radio frequency (RF), reverse osmosis (RO),

5
M.A. Farag et al.
slightly acidic electrolyzed water (SAEW), stainless steel (SS), surface micro-discharge (SMD), thermosonication,(TS), (E-cinnamaldehyde (TC), ultraviolet irradiation
(UV), UV based advanced oxidation processes (UV-AOPs) (UV/hydrogen peroxide, UV/H2O2 and UV/peroxymonosulfate, UV/PMS), UV-C light emitting diodes
(UV–C LEDs), ultrafiltration (UF), ultrasonication (US), ultraviolet light emitting diodes (UV-LEDs),vancomycin-loaded spore targeting iron oxide nanoparticle (van-
IONP), Water activity (aw).
Fig. 3. Spores’ inactivation methods and its application in different food matrices.
used for pasteurization or sterilization of liquid food such as milk or
apple juice (Fan et al., 2019; Lv et al., 2019).
Pretreatment with ultrasound prior to thermal application produced
microbubbles, cavitation, and resulted in capturing the generated heat
to better inactivate Bacillus subtilis spores in water, milk, and rice
(Fig. 3). However, the combination had a slight effect to decrease the
time of decimal reduction (D value) asides from not being available yet
commercially (Ansari, Ismail, & Farid, 2017). Different log reduction
was achieved for different spores i.e., bacteria and fungi, suggestive of
no universal set parameters for inactivation of all microbial spores and
moreover complicated by the different food matrices they are in. Further
studies are needed to evaluate the effectiveness of these combinations on
fungal spores that might present in food.
Combining ultraviolet (UV) radiation with heat decreased the time
needed to inactivate B. subtilis spores in milk. Although with the limited
transmittance of milk due to fats and total solids, UV and heat together
can act as an alternative inactivation method. Results of exposure for 30
s at 110 ◦ C accomplished 6 and 3 log reduction in skim and whole milk of
bovine, respectively (Ansari, Ismail, & Farid, 2019). Higher log reduc­
tion was achieved in skim milk to encompass lower total solid content
and higher penetration capability than whole milk.
Tomato juice inoculated with 1.7 × 108 CFU/mL of B. coagulans
6
Trends in Food Science & Technology xxx (xxxx) xxx
Fig. 2. Schematic diagram showing sequen­
tial heat treatment effect on bacterial and
spores’ count. The diagram shows the effect
of sequential heat treatment impact on bac­
terial and spores counts. In the first heating
step, germination of spores and decrease of
bacterial counts may be promoted. Further
heat application may also inactivate the
previously germinated spores. For bacterial
total count enumeration, tryptic soy agar
(TSA) media can be used, while plate count
skimmed milk agar (PCSMA) can be used for
spores’ tests.
spores exposed to 600 Mpa for 30 s at 85, 95, 100, and 105 ◦ C showed
reduction in the initial count by 1.5–3.4 log reduction, while less than
10 CFU/ml was observed upon 10, 7, 4 and 3 min and 40 s of pressure
holding time at 75, 85, 95, 100 and 105 ◦ C, respectively. The same re­
sults were achieved with stand-alone heating, though at high tempera­
ture and longer time of exposure of 13 and 4 min at 100 and 105 ◦ C,
respectively (Daryaei & Balasubramaniam, 2013). The less time of heat
exposure is likely to preserve tomato juice nutrient value and highlight
for its added value in spore’s inactivation.
Benzalkonium chloride (BAC), an antimicrobial with no effect on
spores alone when combined with moderate heating and slightly acidic
electrolyzed water (SAEW) at 60 ◦ C led to 1.9 log reduction of B. cereus
for a 10 min exposure. The free Cl2 level and time of exposure are two
factors that affected the inactivation magnitude. Consequently, without
the addition of BAC, a lower inactivation level was recorded of 0.7 and
0.6 log for the same ATCC sequence. Such a combination provides a high
cleaning level on solid surfaces that has contact with food (Hussain,
Tango, & Oh, 2019).
1.3. Pressure
HPP, high-pressure processing enhanced the thermal deactivation of
M.A. Farag et al.
milk inoculated with B. cereus spores. At HPP, an increase of thermal
temperature from 38 to 70 ◦ C reduced spore count by 3.5 log reduction.
However, higher specific energy was needed to reach 5 log reduction
with around 400 kJ/L (Evelyn & Silva, 2015), to negatively affect milk
quality.
(E)-cinnamaldehyde (TC) combined with high pressure (HP)
destroyed B. cereus spores in reconstituted powdered infant formula
(RPIF) with 3 log reduction at 0.1% of TC and 600 MPa. This method
was applied at room and refrigerator temperatures according to CDC
storage times. Although the enhancement of microbiological safety and
storage with no change in sensory characters, TC imparted a cinnamon-
like taste to powder (Cetin-Karaca & Morgan, 2018), which might be
disfavored upon ingestion.
1.4. UV radiation
A mixture between UV and iodine gas (I2 g), at 4 ppm per chamber
was used to inactivate Bacillus thuringiensis var. kurstaki (Btk) spores
collected from heating, ventilation, and air conditioning (HVAC) system
high-efficiency filters (Table 1). This blend demonstrated a potent
deactivation effect on spores. A synergistic effect for that mixture was
revealed as employing UV followed by iodine fumigation showed better
deactivation synergism than applying them together at the same time or
the opposite arrangement (Nakpan, Yermakov, Indugula, Reponen, &
Grinshpun, 2019), this technique has potential to be used in surfaces
fumigation and HVAC systems.
UV light with emitting diodes (UV-LEDs) is used as an alternative for
the eradication of water fungal spores i.e., Aspergillus niger, Penicillium
polonicum, Trichoderma harzianum. UV-LED showed higher inactivation
effect over low-pressure UV at 254 nm, however, high electric con­
sumption was consumed by UV-LED. The mechanism by which UV-LED
radiation inhibited photo reactivation was mediated via the destruction
of spore membrane and the increase of intracellular reactive oxygen
species (Wan et al., 2020). Although the method tackled fungal spores in
water, further investigation should be carried out for its effect on
foodstuff and bacterial spores’ inactivation.
Using UV or combined with advanced oxidation processes (AOPs)
such as; UV/hydrogen peroxide (UV/H2O2) and UV/peroxymonosulfate
(UV/PMS) demonstrated an effective response on water chlorine resis­
tant spores i.e., B. cereus (Table 1). The inactivation rate was directly
proportional to UV dose as increase in UV dose to 180 mJ/cm2 led to a
higher log reduction of 3. The UV dose was also further decreased by
adding AOPs to 120–140 mJ/cm2. The inactivation mechanism was
mediated via spore cell membrane and cytoplasm destruction causing
the release of intracellular components. Comparing among the three
aforementioned treatments, the highest inactivation rate was achieved
by UV/PMS, followed by UV/H2O2 versus lowest for UV without AOs
(Zeng et al., 2020).
Moreover, UV/PMS can be used to disinfect fungal spores in drinking
water. UV/PMS exposure led to spores cell wall and membrane damage
concurrent with the release of intracellular structures and the shrinkage
of fungal spores by the effect of the generated reactive radicals (Wen, Xu,
Zhu, Huang, & Ma, 2017). Using UV/PMS should be evaluated in terms
of safety and quality aspects as these radicals formed in the medium can
affect the container and or product.
Pretreatment disinfection of water by adding chlorine synergistically
enhanced UV irradiation. The addition of 4 mg/L of Cl2 resulted in an
increment of 1.8, 1.5 log in UV265/Cl2, and UV280/Cl2, respectively.
UV265/Cl2 showed a slightly higher inactivation rate constant than
UV280/Cl2 (G.-Q. Li, Huo, Wu, Lu, & Hu, 2018). Radio wave frequencies
were applied to inactivate B. cereus spores in a powder of red pepper.
Contamination of herbal spices with spores represents a challenge as
they are often used raw with no heat processing evading to be dis­
infected using detergents. Heating radio frequencies achieved 4 log re­
ductions at 90 ◦ C temperature for 110 s followed by 12 min holding time
with a water activity of 0.7. Such a technique has the potential to be
7
Trends in Food Science & Technology xxx (xxxx) xxx
applied with some modifications to control spores in food with low
moisture content (Jiao, Zhang, Hu, & Zhao, 2019). Such technology can
be used in food that contains polar groups as liquids, water and milk, and
yogurt pasteurization.
A newly developed hurdle technology of microwave-combined cold
plasma treatment (MCPT) effectively decontaminated B. cereus spores in
red pepper, especially at high microwave power. Pretreated red pepper
with drying vacuum achieved more spore’s reduction than that using
drying IR. IR caused surface cracks and crevices for red pepper which
might be disfavored by consumers. Higher spores inactivation was
achieved at high microwave power with an increase of water activity to
reach 2.6 instead of 1.7 reduction log (Table 1) as a consequence of the
dissociation of hydrogen bond in water generating OH− radical that kills
bacteria, and with no effect on the red pepper color by MCPT (Kim, Choi,
Lee, & Min, 2017).
Exposure to microwave (Table 1) inactivation effect on spore was
compared to that of conductive heating of spores for a similar time
resulting in the eradication of 107 colony forming units (CFUs) in the
case of 800 W UV irradiation and not observed with conductive heat for
1 min exposure, suggestive that such non-thermal method is a simple
and time-efficient technique for spore’s removal. This exposure was
studied on Clostridium difficile spores which can be found in several food
products such as retail meat products (Ojha, Chankhamhaengdecha,
Singhakaew, Ounjai, & Janvilisri, 2016).
1.5. Pulsed light
Application of pulsed light (PL) to deactivate both vegetative and
spore forms of B. subtilis and Geobacillus stearothermophilus was depen­
dent on two factors, cell density through which transmission of light
occurred and the initial count of the bioburden. Lower inactivation level
was observed after PL exposure, though with spores found more resis­
tant than vegetative forms to PL (Artíguez & Martínez de Marañón,
2015).
Another non-thermal procedure for spore’s inactivation is intense
pulsed light (IPL). This method strongly inhibited B. subtilis spores with a
net effect of 7 log reduction when 7.4 J/cm2 of IPL was applied
(Table 1). Being a non-thermal technique, it can be safely implemented
in food products (Jo, Hwang, & Chung, 2019), without much affecting
its food value. Though such hypothesis needs to be further confirmed
using biochemical analysis of food components or simple bioassays i.e.,
antioxidant effects.
1.6. Plasma
Another cold method to inactivate fungal spores is non-thermal
plasma (NTP) using a rotating reactor. The targeted spores were Asper­
gillus sp. in dried granular spice i.e., black pepper (Fig. 3). The inocu­
lated count of spores showed 3 logs reduction upon 4 min treatment.
Moreover, the addition of humidified gases enhanced the inactivation
process to produce no viable spores after similar time of treatment.
Although, it should be noted that black pepper nutritive quality post
treatment was negatively impacted with lower antioxidant capacity than
dry heat-treated peppers, with no change in piperine alkaloid level
(Tanino, Arisaka, Iguchi, Matsui, & Ohshima, 2019). Assessment of food
quality post spore’s inactivation should be implemented in other ap­
proaches to allow for comparison of both aspects that is spores inacti­
vation power while maintaining food quality targeting both fungal and
bacterial spores.
The cold plasma sterilization is a new technology in which reactive
ionized neutral gases deactivate both spores and bacteria in food ex­
amples i.e., black pepper, rice, and meat, without affecting food physi­
cochemical properties. The lethality resulted from the generation of O3
gas reactive species. Moreover, an additional advantage, the exact pre­
diction of sterilization time could be estimated for sterilization cycle
parameters (Mendes-Oliveira, Jensen, Keener, & Campanella, 2019).
M.A. Farag et al.
Plasma activated water (PAW), as a disinfectant, when inoculated
with 106 CFU Ml− 1 showed 1.6–3 log reduction of B. cereus at 55 ◦ C
(Table 1). Several factors positively impacted the performance of PAW,
such as increased temperature, a lower initial count of spores, and
smaller PAW volume (Bai et al., 2020).
1.7. Chemicals
The disinfectant sporicidal function of para-acetic acid (PPA) or
H2O2 as gas was compared to their liquid state. The comparison was
tested against the inoculum of highly resistant spores of
G. stearothermophilus. Both states of PPA achieved 5 log reductions in 10
min for gas versus 4.5 min for liquid which indicates the higher effect of
disinfection for liquid from. However, the fog form has fine small droplet
size which permits decreased dose and cost-plus improved compatibility
with food material under disinfection. Fumigation with PPA 0.06%
inactivated spores efficiently, whereas 12% H2O2 solution achieved
close results while its vapor application took 60 min to result in 3 log
reduction (Table 1) (Hayrapetyan, Nederhoff, Vollebregt, Mastwijk, &
Nierop Groot, 2020).
Fungal disinfection by chlorine dioxide ClO2 can kill fungal spores in
water (Fig. 3). ClO2 amount and temperature affected inactivation
positively while humic acid and water negatively impacted the disin­
fection process. In comparison with Cl2, ClO2 exhibited a higher
oxidation potential and increased cell membrane permeability leading
to leakage of the intracellular component from spores’ cell membrane
and wall destruction. However, due to its instability and degradation at
higher levels, ClO2 generation must be done on-site, (Wen, Xu, Huang,
Zhu, & Ma, 2017).
Surfactants such as poly-L-lysine, thiamine dilaurylsulfate, and tor­
ilin effectively achieved 1.5 to 2.5 logs reductions of Clostridium. spor­
ogenes fungal spores at 1–2% dose levels. These chemicals are
amphipathic to denature spores coat protein by binding to protein coat
with surfactants’ hydrophobic and hydrophilic moieties as in soap.
Stronger effectiveness was observed using more hydrophobic surfactants
such as poly-L lysine, thiamine dilaurylsulfate, and torilin (Cho &
Chung, 2018). Trials should be further carried out to study the effect of
these several surfactants on variety of food such as dried herbs versus
water rich food.
Enterocin, AS-48, a cyclic peptide produced by E. faecalis. treatment
inactivated G. stearothermophilus endospores rapidly in canned corn and
peas and coconut milk. Moreover, enterocin AS-48 disrupted the
permeability of the cellular membrane of vegetative bacteria, concur­
rent with an increase in endospores sensitivity against heat. Despite it
was found ineffective towards intact endospores. Trypsin addition
negatively prevented enterocin activity and increased the survival of
G. stearothermophilus after short time of enterocin AS-48 exposure (Pilar
Martínez Viedma et al., 2009).
Another enterocin, EJ97 produced by Enterococcus faecalis EJ97
treatment inactivated G. stearothermophilus spores added to canned food
products. After 5 min treatment, dormant endospores showed though
resistance. An antimicrobial response was increased by combining
enterocin EJ97 with heat at 90 and 95 ◦ C suggestive for the advantage of
hurdle technology in spores inactivation (P. M. Viedma, Abriouel, Ben
Omar, Lopez, & Galvez, 2010).
1.8. Pulsed electric field
Pulsed electric field (PEF) was used to inactivate B. cereus spores in
milk (Table 1). Different responses upon exposure were observed such as
inactivation resistance to PEF alone, negative effect when combined
with heating at 40 ◦ C versus inactivation at 50 ◦ C. The study showed
better results for skim milk inactivation compared to whole milk likely
due to less matrix interference or might be different interaction mech­
anism. When nisin (a polycyclic antibacterial peptide) was added, 3.6
logs reductions were achieved. Even after applying several preservation
8
Trends in Food Science & Technology xxx (xxxx) xxx
parameters, the limitation of this study was still spore resistance,
achieving at the best 3.6 log reduction (Bermudez-Aguirre, Dunne, &
Barbosa-Canovas, 2012).
1.9. Photocatalism/photosensitization
Bacterial spore photocatalytic deactivation was enhanced by the
deposition of TiO2 films with a silver (Ag) metal and UV radiation. After
UV-A radiation of B. subtilis, a significant rise in the photocatalytic
inactivation was recorded by increase in Ag content. This increase
reached the highest efficiency at 1 wt % of Ag/TiO2. However, adding
excess amounts of Ag caused a decrease in the inactivation as a correct
ration is required to achieve the inactivation (Zacarías, Satuf, Vaccari, &
Alfano, 2015).
A fabricated electrode with TiO2 nanoparticles with activated C fiber
is used in an all-solid-state photoelectrochemical cell (ASPEC) to photo
inactivate Penicillium expansum fungal spores. The cell reduced spores’
number by 1 log photo electrocatalytically and to be considered for the
inactivation of spores in food cold storage areas (Su et al., 2020). A
polyphenolic natural product curcumin found in curcuma, when com­
bined with visible light at a wavelength of 420 can produce up to 3, 2 log
reduction of Aspergillus flavus fungal spores. in suspension and maize
kernels, respectively. This effective decontamination method is medi­
ated though curcumin photosensitization effect (Temba, Fletcher, Fox,
Harvey, & Sultanbawa, 2016).
1.10. Miscellaneous
Clean in place (CIP) system of (NaOH and HNO3) effectively elimi­
nated C. perfringens spores, which can adhere to stainless steel surfaces
for almost 2 days. Spores coat exhibits a hydrophobic character which
aids in its adhesion. This system can prevent cross-contamination of
these spores in the food industry across surfaces (Alzubeidi, Udompi­
jitkul, Talukdar, & Sarker, 2018), but cannot be applied for food treat­
ment due to acid and alkali corrosive effects.
Comparing hyperbaric storage to room and refrigerating tempera­
tures and atmospheric pressure for a month, 105 CFU mL− 1 Alicycloba­
cillus acidoterrestris spores’ inoculum apple juice showed deactivation to
less than 10 CFU/mL upon exposure to 50 and 100 MPa pressure at
ambient temperature, whereas spore initial count did not change under
refrigeration for a 30-day duration. These results pose hyperbaric stor­
age an optimum deactivation condition in acidic food type as in apple
juice during a month of storage (Pinto et al., 2019). Longer storage
period interval studies should be tested in the future for efficacy
measurement.
2. Spores borne food detection methods
Many procedures are followed in the food industry to eliminate mi­
crobial contamination or growth that may lead to its spoilage, foodborne
illness, or even death. The resistance of spores to inactivation poses
detection as an indispensable tool as important as microbial detection
for health, economic, and environmental aspects. In the upcoming sec­
tion, we will discuss the different spore detection methods in the liter­
ature highlighting its applications in which food types, advantages, and
limitations.
2.1. Chromatographic, microscopic & spectroscopic methods
To identify a fingerprint for each pathogenic spore is an added value
in food quality control measures (Fig. 4). Fingerprints can be a cell
component i.e., fatty acids, dipicolinic acid (DPA), etc. Whittaker et al.
demonstrated that spores of B. anthracis and B. cereus can be identified
rapidly by their different fatty acids profile without prior germination.
For instance, the fatty acid iso 17:1 ω10c is present in B. cereus spores
and not in B. anthracis. While B. anthracis spores have a profoundly
M.A. Farag et al. Trends in Food Science & Technology xxx (xxxx) xxx

Fig. 4. Bacterial spore structure, detection targets in spore cells, and suitable detection methods targeting each cellular part.

higher content of anteiso fatty acid (branched-chain fatty acids) profile
consistent with 15:0, 17:1, and 17:0 compared to B. cereus spores. Gas
chromatography (GC) coupled to flame ionization detector provided a
comparison of fatty acid profiles in spores suggesting that spores fatty
acids full profile can be used as a fingerprint, however, spores’ germi­
nation might be needed still to confirm its identification (Whittaker
et al., 2005).
Although contrast phase microscopy has the advantage of observing
endospore’s morphology without prior staining or germination
(Table 2), it suffers from false detection i.e., air bubble as a living or­
ganism. Consequently, a complementary confirmatory tool such as
spores culturing on a specific media is needed. Such limitations make it
rather inconvenient for large scale analysis in food specimens (Guine­
bretiere, Girardin, Dargaignaratz, Carlin, & Nguyen-The, 2003).
Combining two techniques can help reduce the limitations of one
another (Table 2) and confirm its identification. By using scanning
electron microscope (SEM) for morphological identification of A. flavus
spores on the surface of edible locust in parallel to high-performance
liquid chromatography coupled to fluorescence detection (HPLC-FLD)
for mycotoxin assessment, a convenient method presented for large scale
fungal spore screening of edible or medicinal animal matrices (Kong,
Kong, Yang, & Yang, 2018).
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a
surface spectroscopy method that identifies surface cellular components
such as phospholipids and fatty acids (Fig. 4). Thompson et al. used it as
a comparative tool of surface phospholipids between spores and vege­
tative cells of B. megaterium. Phospholipids such as diglyceryl diac­
ylglycerols and cardiolipins were more abundant in the spore form than
the vegetative form. These phospholipids possess higher boiling points,
with a major reduction in fatty acid side chains unsaturation and to

Table 2
Microscopic and spectroscopic methods for the detection of spores in various foodstuff.
Detection method Microorganism/strain Food type Advantages Disadvantages References

Contrast phase microscopy B. cereus Zucchini puree Simple, easy, fast, portable Guinebretiere
et al. (2003)
Combined SEM for spores in A. flavus Medicinal and edible low cost, sensitive, rapid, and SEMs: limited to solid Kong et al. (2018)
parallel to HPLC-FLD for locust robust samples, difficult setup, need
mycotoxin skilled personnel.
HPLC-FLD: post column
derivatization
Capillary gas chromatography B. anthracis, B. cereus N/A Sensitive, specific, fast, low cost Sample destruction Whittaker et al.
(GC) with flame ionization (2005)
Raman spectra and SERS B. thuringiensis kurstaki N/A Minimal sample manipulation, Normal Raman: Low Morrow et al.
ATCC 33679 rapid, robust. enhanced signal, sensitivity (2012)
reproducible, sensitive
SERS B. cereus, B. subtilis N/A Enhanced signal, fast, Cowcher et al.
reproducible, sensitive, portable (2013)
Raman spectroscopy B. anthracis Household powders Specific, non-destructive Low sensitivity Stockel et al.
(baking powder, milk (2012)
powder)
Micro-Raman spectroscopy B. anthracis, B. subtilis B. N/A Fast, sensitive, low cost, minimal Stockel et al.
mycoides sample preparation, single (2010)
B. sphaericus, endospore level
B. thuringiensis
MALDI-TOF-ICMS B. anthracis, B. cereus, B. N/A Simple, fast, minimal sample Incompatible with destructive Lasch et al.
cereus, B. licheniformis, preparation spore inactivation methods (2008)
B. subtilis, B. thuringiensis
TOF-SIMS B. megaterium DSMZ 32 N/A Sensitive, fast, minimal sample Thompson et al.
preparation (2004)

N/A: Food type is not mentioned., SEM: Scanning electron microscope, HPLC-FLD: High-Performance Liquid Chromatography with Fluorescence Detection, SERS:
Surface-enhanced Raman spectroscopy, DPA: Dipicolinic acid, CaDPA: Calcium dipicolinate, MALDI-TOF-ICMS: Matrix-assisted laser desorption/ionization – Time of
flight-Intact Cell Mass spectrometry, TOF-SIMS: Time of flight- Secondary-ion mass spectrometry.

9
M.A. Farag et al.
provide spore its surface resistance against surrounding conditions
(Thompson, Jungnickel, Lockyer, Stephens, & Vickerman, 2004). With
the advances in MS based profiling techniques of lipids and moreover to
be coupled with multivariate data analysis, better discrimination of
spores from different genotypes can be achieved in the future. The
application of chemometric classification tools has caused a paradigm
shift in biological specimens analysis (M. A. Farag et al., 2016) and has
yet to be fully exploited in microbiological analysis.
Raman spectroscopy, a vibrational spectroscopy method, showed
promising results in the detection of bacterial spores. Stockel et al.
distinguished B. anthracis spores from different Bacilli strains in different
environmental and household powders (Table 2) based on the unique
calcium dipicolinate salt (CaDPA) of its spore and DPA spectral char­
acteristics. With minimal sample preparation, satisfactory results were
obtained, detection accuracy ranged from 91.2 to 100% with 99.6% for
B. anthracis (Stockel, Meisel, Elschner, Rosch, & Popp, 2012). Morrow
et al. further employed Raman spectroscopy to distinguish between
different physiological forms of B. thuringiensis and compared it to
surface-enhanced Raman spectroscopy (SERS) as methods for initial
screening of bio-threat in food and water. Improved resolution was
achieved by applying SERS to overcome the noise resulting from any
fluorescent particle in the food matrix. By using the metal interface in
SERS i.e., silver (Morrow, Almeida, Cole, & Reipa, 2012), or colloidal
silver (Cowcher, Xu, & Goodacre, 2013), not only it enhanced the signal
to noise ratio but rather showed improved reproducibility. The sensi­
tivity of SERS reached a level of 5 ppb (29.9 nM) for DPA detection in
B. cereus and B. subtilis among other Bacillus species. This sensitivity can
reach 0.1 ppb but requires the use of a Raman microscope, which
compromises the portability of the method (Cowcher et al., 2013). While
in micro Raman spectroscopy, light is focused on the sample through a
microscope, reducing interference from the matrix due to the small
sample region detected. Another advantage elaborated by applying the
micro Raman spectra other than the feasibility of the on-site diagnosis
lies in the compatibility to detect spores after their inactivation by
formaldehyde (Stockel et al., 2010). Profiling of spore’s metabolites
composition can help identify further chemical targets to be used for its
selective detection in the future as observed in case of CaDPA.
2.2. Biosensors
Biosensors, usually named “Lab on a Chip” are convenient on-site
detection methods for their small size, portability, ease of use and do
not necessarily require skilled personnel to operate. They also provide a
tentative screening before applying other sophisticated techniques;
response is based on a signal that is directly proportional to the analytes’
concentration. A typical biosensor is composed of the analyte, substance
of interest to be detected i.e., spores, glucose, etc.; bioreceptor, the
substance the binds specifically to the analyte for recognition i.e., whole-
cell, enzyme, protein, antibody; transducer, part responsible for con­
verting the biorecognition signal into a measurable form, and finally
electronics and display responsible for signal amplification and visual­
ization (Bhalla, Jolly, Formisano, & Estrela, 2016).
With regards to biosensor application for spore detection, Yang et al.
presented a convenient method for the early spore detection of the rice
blast caused by Magnaporthe oryzae fungus to the rice crop. The pro­
posed device was relatively cheap, portable, and with a 5.9% error rate.
Complementary metal-oxide-semiconductor (CMOS) offered a wider
imaging area of 20 mm2 that is robust and not affected by external
factors i.e., humidity, temperature. The microfluidic chip is designed to
filter airborne impurities from the spores thus decreasing impurities
interference. Spores enrichment takes place on the CMOS area to allow
lens less diffraction fingerprinting afterward and of potential to be
employed for the onsite detection for other fungal spores (N. Yang et al.,
2019). A highly sensitive paper-based immuno-sensor was employed for
the early spore’s detection of the Asian soybean rust caused by Pha­
kopsora pachyrhizi fungus to the soybean crop. By using high binding
10
Trends in Food Science & Technology xxx (xxxx) xxx
nitrocellulose as a membrane for spores binding, along with polyclonal
antibodies and fluorescent nanoparticles for bio recognition, a limit of
detection (LOD) of 2.2 ng/ml was achieved. Such biosensor can be used
by unskilled workers using a UV lamp for excitation and naked eye
recognition of the fluorescence, and with detection limits comparable to
complex ELISA and PCR assays (Miranda, Linares, Thalhammer, &
Kubota, 2013).
A less sensitive bead-based sandwich hybridization system was uti­
lized with an electric chip for the detection of B. cereus spores after its
disruption based on endosporal DNA. The signal strength was directly
proportional to the hybridization efficiency, with the larger the DNA
target, the less its mobility and harder to get in contact with the probe.
The target sequence position affected the efficiency as well as the rela­
tively high sensitivity level of as few as 107 spores (M. Gabig-Ciminska,
H. Andresen, J. Albers, R. Hintsche, & S. O. Enfors, 2004).
Micromechanical cantilever array was used for immobilization,
germination, and detection of Aspergillus niger and Saccharomyces cer­
evisiae fungi. Three different protein coatings were compared to include:
concanavalin A (Con A) and fibronectin (Fn), based on their affinities to
different surface glycoproteins in spores, and anti-Aspergillus niger poly-
clonal antibodies (IgG). Detection levels reached 103–106 CFU/ml with
IgG coating showing the best binding efficacy for immobilization (IgG >
Con A > Fn) (N. Nugaeva, Gfeller, Backmann, Lang, Duggelin, et al.,
2005).
Electrically active polyaniline coated magnetic (EAPM) nano­
particle, a novel transducer, was used to detect the bacterial spores of
B. anthracis in different food samples (Table 3). By using sandwich
immunoassay method, LOD reached 4.2 × 102 spores/ml in some
matrices (lettuce and ground beef) and 4.2 × 103 spores/ml in whole
milk. The nanoparticles were conjugated with IgG for B. anthracis to
allow easy magnetic separation from the used complex food matrices
(Pal and Alocilja, 2009).
Geosmin, a volatile product of Streptomyces species, was used as a
marker for the early detection of spores in potable water. By using an
electronic nose (e-nose) consisting of an array of 14 conducting polymer
sensors, different volatiles pattern allowed for the differentiation be­
tween S. aureofaciens and S. griseus with a high sensitivity level of 102
spores/ml (A. C. Bastos & Magan, 2006). Despite the advantages and
limitation in this application (Table 3), due to complexity and variability
in environmental samples, it opens the door for more detection targets
(Fig. 4) based on volatile products profiling, Such approach was applied
successfully with various applications in plant specimens (M. Farag,
Hegazi, Dokhalahy, & Khattab, 2020).
A sandwich enzyme-linked immunosorbent assay (ELISA) was
employed for the detection of B. globigii spores in environmental and
industrial water samples, without prior enrichment, with an anti-
B. globigii. Detection range reached 10 spores/sample independent of the
sample size but has a relatively low upper limit (103-105 spores) with
respect to the sample medium and flow rate. Different water samples’
pH were studied (7.2–9.2), with alkaline pH to denature antibodies
causing signal drop off among other limitations (Table 3) (Weimer et al.,
2001).
2.3. Immunoassay
Spores of B. anthracis (Wang et al., 2015) and B. stearothermophilus
(Blake & Weimer, 1997) were both detected by immuno-magnetic
capture technique. Lateral flow immunochromatographic assay (LFIA)
was employed for B. anthracis detection based on the blockage to the
chromatographic pores by the immune complex conjugate, thus forming
visible retention lines that can be detected by naked eyes, optical or
magnetic signal analysis. Spores detection level was high in different
powdery matrices without prior treatment, i.e., milk powder (6 × 104
spores/g), starch (2 × 105 spores/g), and baking soda (5 × 105 spores/g)
(Wang et al., 2015). While in case of B. stearothermophilus,
biotin-avidin-amplified sandwich ELISA was coupled to fluorescence
M.A. Farag et al. Trends in Food Science & Technology xxx (xxxx) xxx

Table 3
Biosensors determination of spores in foodstuff.
Detection method Microorganism/strain Food type Advantages Disadvantages References

Microfluidic chip and Magnaporthe oryzae Rice Cheap, mini size, fast, on-site, early (Ning Yang et al., 2019)
lens-free diffraction (rice blast fungus) detection
Fingerprint
Paper-based immuno- Phakopsora pachyrhizi Soybean Highly sensitive, disposable, on-site & (Miranda et al., 2013)
sensor (Asian soybean rust) early detection, accurate, specific, fast,
does not need skilled personnel
(EAPM) nanoparticle- B. anthracis Lettuce, ground Fast, Sensitive, easy to use Low sensitivity (Pal and Alocilja, 2009)
based biosensor beef, and whole
milk
E-Nose Streptomyces Potable water Fast, reproducible (Bastos and Magan, 2006)
aureofaciens, S. griseus
Micromechanical Aspergillus niger, N/A Fast, precise, specific Requires clean (Natalia Nugaeva et al.,
cantilever arrays Saccharomyces environment, low 2005)
cerevisiae sensitivity
Electro-chemical chip B. cereus B. subtilis, N/A Specific, cheap Time is dependent on (Gabig-Ciminska, Andresen,
B. thuringiensis LOD Albers, Hintsche, & Enfors,
2004)
SELEX Alicyclobacillus Orange juice Cheaper than antibody, rapid, sensitive Low selectivity (Hünniger et al., 2015)
acidoterrestris simulating buffer
Immuno-flow/ELISA B. globigii Different types of Highly sensitive, rapid, specific Several dilutions are (B. C. Weimer, Walsh, Beer,
water needed to fit into Koka, & Wang, 2001)
detection range

CMOS: Complementary metal-oxide semiconductor, EAPM: Electrically active polyaniline coated magnetic, N/A: Food type not mentioned, LOD: Limit of detection,
SELEX: Systemic evolution of ligands exponential enrichment.

quantification to achieve a higher LOD of 8 × 103 CFU/ml in
ultra-high-temperature pasteurized (UHT) skimmed milk among
different food matrices (Table 4), (Blake & Weimer, 1997).
Catalyzed reporter deposition - fluorescence in-situ hybridization
(CARD-FISH) and in-situ polymerase chain reaction (PCR) were used as
signal amplifiers in the detection of antibiotic-resistant genes in B. cereus
spores. Permeabilization while maintaining the spore integrity to pre­
vent the loss of cell components is considered a key factor in this tech­
nique. CARD-FISH was able to detect the high copy number (pC194)
plasmid only while in-situ PCR showed higher sensitivity for the detec­
tion of both the low and high copy number plasmids (pC194,
pMTL500Eres). This method presents a promising, time-efficient for
specific elements detection in spores based on single-cell analysis that
can be examined for the detection of other Bacillus spp. spores
(Laflamme et al., 2009).
The detection of B. anthracis spores in complex matrices i.e., milk
products, along with staphylococcal enterotoxin B and clostridial bot­
ulinum neurotoxin A simultaneously were achievable by using electro­
chemiluminescence (ECL). Bacillus anthracis is the agent of anthrax—a
common disease of livestock and, occasionally, of humans and of value
for its detection in both food and in bioterrorism. By applying a sand­
wich immunoassay using improved MAb against surface antigens of the
spore enhanced the sensitivity to reach LOD of 4 × 104 CFU/ml (Sach­
deva et al., 2014). Another luminescence assay method was applied for
the detection of Bacillus spp. spores in different matrices (Table 4). By
targeting the sporulation gene (spo0A) post-heat activation, a
filtration-based ATP bioluminescence detection was conducted within
20 min with ATP signal amplification, which is significantly less time
consuming than other methods. LODs obtained were as low as 1.4 × 102
and 1 × 103 CFU/cm2 for B. amyloliquefaciens and B. licheniformis spores

Table 4
Immunoassay based determination of spores in food.
Detection method Microorganism/strain Food type Advantages Disadvantages References

Super-paramagnetic B. anthracis A16 (pXO1þ, pXO2þ) Milk powder, starch, and Sensitive, cheap, Low Wang et al. (2015)
LFIA baking specific, naked-eye sensitivity
Soda detection
Immuno-magnetic ELISA B. stearothermophilus UHT skimmed milk, fluid Minimal sample Low Blake and Weimer
milk, powdered milk, baby preparation, specific sensitivity (1997)
formula, spices, soil, and
sand
CARD-FISH and in-situ PCR B. cereus N/A Rapid, sensitive, Time- Laflamme et al.
coupling with PCR consuming (2009)
detects low copy
numbers
ELISA Aspergillus versicolor, Cladosporium Cottage cheese and yogurt Nonspecific ELISA can Low Yong and Cousin
herbarum, Fusarium poae, Geotrichum detect general mold sensitivity (1995)
candidum, Mucor circinelloides, and contamination of foods
Penicillium chrysogenum
Electrochemiluminescence assay B. anthracis Dairy milk products (raw Highly sensitive, rapid, N/A (Sachdeva, Singh, &
and whole milk, (UHT) robust, and multiplex in Sharma, 2014)
milk, skim milk, cream nature
milk, and chocolate milk)
Filtration-based ATP B. amyloliquefaciens, B. licheniformis, B. Non-alcoholic beverage Rapid and quantitative N/A Ratphitagsanti, Park,
bioluminescence and real-time thuringiensis, packages and food powders Lee, Wu, and Lee
PCR ABTS-351 (garlic & onion powders, (2012)
creamy potato soup mix)
Streptavidin-Conjugated Single- B. cereus N/A Sensitive N/A Koo, Foegeding, and
Chain Antibody Swaisgood (1998)

11
M.A. Farag et al.
on food packages (Polyethylene Tere-phthalate film) respectively.
Whereas, for B. thuringiensis spores in food powder, LODs were at 7.9 to
3.2 × 104 CFU/mg (Ratphitagsanti et al., 2012).
2.4. Molecular/protein-based detection of spores in food
Several molecular techniques have been reported for detecting bac­
terial and fungal spores in food, though with differences in sensitivity
level and accuracy. In this section, we provide a summary of molecular
approaches for spores’ detection in food and beverages.
2.4.1. Gel free proteomic
This method is based on detecting and identifying proteinaceous coat
and the exosporium layer in spores (Abhyankar et al., 2013), that are
vital in spores pathogenicity, mitigating against inactivation techniques
and to aid for its survival in food (Abhyankar et al., 2013), and are thus
considered good targets to be used for spores detection. By determining
the conserved proteins unique for each species, rapid, sensitive, and easy
detection of spores can be thus achieved. Gel free proteomics has been
applied for the detection of Bacillus spp (B. cereus, B. subtilis) and Clos­
tridium difficile as a rapid and sensitive spores detection method
(Abhyankar et al., 2011; Abhyankar et al., 2013). Nevertheless, the
targeted protein is detected based on the sequence of a single tryptic
peptide which is not enough specific as post-translation modifications
can yield various protein products, and with the differentiation between
all of these protein products by analyzing single tryptic peptide from the
derived protein is impractical (Baggerman, Vierstraete, De Loof, &
Schoofs, 2005) (Fig. 5) (Suppl. Table S1). Comprehensive protein
profiling of microbial spores may aid in the identification of specific
peptide regions for its detection, especially with the advances in prote­
omics field (Graham, Graham, & McMullan, 2007).
Fig. 5. Molecular methods developed for detecting fungal and bacterial spores in various foodstuff.
12
Trends in Food Science & Technology xxx (xxxx) xxx
2.4.2. Polymerase chain reaction (PCR)
In contrast to peptide-based recognition of spores, PCR is based on
detecting the targeted gene or DNA sequence in each microorganism.
The specific DNA fragments for each microorganism can be then
amplified using PCR(Mullis, 1990). In the food industry, PCR provides
considerable sensitivity and rapid detection of microbial spores. For
instance, in, Alternaria sp. and Fusarium sp., mycotoxins were detected
based on18S rDNA barcode while in Thermoanaerobacter, Thermoa­
naerobacterium, Moorella, and Caldanaerobius, detection of spores were
based on 16S rDNA sequence (Al Husnain & AlKahtani, 2019; Aoyama &
Miyamoto, 2015), (Fig. 5) (Suppl. Table S1). Although PCR has been
known for its rapid detection of spores, it is limited to qualitative
detection, as a result, a quantitative real-time PCR was developed to
provide both qualitative and quantitative detection methods of spores
(Kubista et al., 2006; Morillo, Lau, Sanz, Herrera, & Silva, 2003).
2.4.3. Quantitative real-time PCR (qRT PCR)
The quantification of contaminants in food by real-time quantitative
PCR has become a common detection method in food microbiology
(Rodríguez-Lázaro et al., 2007). The rapid, specific, accurate, sensitive
detection and quantification method of real time PCR has proved to be
more efficient than conventional PCR (Gil-Serna, González-Salgado,
González-Jaén, Vázquez, & Patiño, 2009). qRT PCR has been used as a
detection method for both bacterial and fungal spores in foodstuffs
(Gil-Serna et al., 2009; Hünniger et al., 2015). In detecting
C. tyrobutyricum, the specific region flagellin gene (fla) which encodes a
protein that involved in bacterial motility has been targeted
(López-Enríquez et al., 2007) (Fig. 5) (Suppl. Table S1). Also, qRT PCR
was able to quantify the number of spores by detecting down to 25
spores in 25 ml of raw or UHT whole milk (López-Enríquez et al., 2007).
2.4.4. Multiplex PCR and multiplex qRT PCR
Multiplex PCR has been utilized to amplify multiple target sequences
M.A. Farag et al.
using multiple primers in a single reaction (Chamberlain, Gibbs, Rainer,
Nguyen, & Thomas, 1988; Elnifro, Ashshi, Cooper, & Klapper, 2000).
This allows for the detection of numerous microorganisms in one reac­
tion (Chamberlain et al., 1988; Elnifro et al., 2000). Consequently, this
method overweight conventional PCR with less effort (Chamberlain
et al., 1988; Elnifro et al., 2000). However, the low sensitivity, speci­
ficity, and the high probability of errors during amplification process
have been documented (Chamberlain et al., 1988; Elnifro et al., 2000).
Multiplex PCR has been applied to screen for BoNT-producing clostridia
in all types A, B, E, and F (De Medici et al., 2009) (Fig. 5) (Suppl.
Table S1). The only difference between multiplex PCR and multiplex
qRT PCR is that multiplex qRT PCR has the advantage of quantification
in addition to detection (Frentzel, Thanh, Krause, Appel, & Mader,
2018). Multiplex qRT PCR method was able to detect and quantify
B. cereus group in spices and herbs with differentiation of closely Bacillus
species (Fig. 5) (Suppl. Table S1) (Frentzel et al., 2018).
2.4.5. New loop-mediated isothermal amplification (LAMP)
LAMP presents an alternative method to PCR for spores’ detection
due to its simplicity. LAMP only needs primer, DNA polymerase, and
reaction mixture, while there is no need for thermocycler as the process
can be conducted in a water bath (Keikha, 2018). In addition, the ability
to detect pathogen spores at an initial infected stage has added a great
value (L. Li, Zhang, & Zhang, 2019). LAMP method detected the fungal
spores of M. oryzae which causes rice blast and destroys rice crop (L. Li
et al., 2019). The quantitative LAMP assay has allowed for the fast
detection (35 min) of the M. oryzae fungal spores at a sensitivity of 3.2
spores per 21 mL by targeting the Alb1 superfamily protein MGG_0432
(L. Li et al., 2019). Also, LAMP has shown great efficiency in predicting
aflatoxin risk in A. flavus, A. parasiticus, and A. nomius (Luo, Vogel, &
Niessen, 2014) (Fig. 5) (Suppl. Table S1). For bacterial spore detection,
LAMP was able to detect T. mathranii and T. thermocopriae spores present
in canned coffee and soft drinks by detecting its 16S rDNA sequence
(Aoyama & Miyamoto, 2015) (Fig. 5) (Suppl. Table S1). However, its
only limitations lie in the lack of commercial kits and the complexity of
its operating mechanism (Keikha, 2018).
2.4.6. Standard mouse bioassay (SMB)
This assay is based on the intraperitoneal (i.p.) injection of infected
samples to mice (Johnson, McAdams-Gallagher, & Aceto, 2016). To
detect C. botulinum, prior to mouse bioassay, a culture method combined
with neurotoxin detection for BoNT-producing clostridia is applied to
allow for spores detection (Johnson, McAdams-Gallagher, & Aceto,
2016). SMB is a rapid, accurate and specific method; however, it is
expensive and time-consuming, and with negative results failing to al­
ways exclude infection (De Medici et al., 2009) (Fig. 5) (Suppl.
Table S1). Compared to multiplex PCR, the major advantage of the
multiplex PCR method that it can detect and easily differentiate BoNT
genes encoding the four neurotoxins types A, B, E, and F associated with
human botulism. Also, multiplex PCR can minimize the number of an­
imal tests conducted by SMB method and are more cheap and rapid in
delivering results (De Medici et al., 2009).
2.4.7. Combined bimodal detection methods
Combining PCR assay with propidium monoazide (PMA) presented a
rapid, specific, and sensitive assay to detect spores i.e., Bacillus spor­
othermodurans (Cattani, Ferreira, & Oliveira, 2013), with a detection
limit of 100 CFU/mL in ultra-high temperature (UHT) treated milk
(Cattani et al., 2013) (Fig. 5) (Suppl. Table S1). Applying Ag/Au as an
electrochemical detection method to the generated qRT PCR products
was able to detect further 2 CFU/ML of spores in A. acidoterrestris with
low detection and quantification limits at the nanomole range (7.07 and
23.6 nM), (Eguiluz et al., 2009) (Fig. 5) (Suppl. Table S1). Another
bimodal detection method involves using an analytical method such as
aptamer-based trapping besides RT PCR in detecting B. cereus spores to
improve the selectivity due to limited aptamer selectivity (Fischer et al.,
13
Trends in Food Science & Technology xxx (xxxx) xxx
2015) (Fig. 5) (Suppl. Table S1). In Alternaria alternata, PCR and ELISA
were used to detect the allergenic protein of A. alternata, Alt a1.
Nevertheless, PCR was found to be more sensitive than ELISA (Gabriel
et al., 2017) (Fig. 5). PCR-color development hybridization assay can
identify Clostridium 16S rDNA sequences in clostridial spores making it a
rapid and sensitive assay (Galindo, Rangel-Aldao, & Ramírez, 1993)
(Suppl. Table S1). Four molecular methods were developed to assess the
spores of Clostridium perfringens (Kaneko et al., 2011). Ordinary PCR,
nested PCR, real-time PCR, and loop-mediated isothermal amplification
(LAMP) were all found able to detect cpe enterotoxin gene in meat
samples with low numbers of cpe-positive C. perfringens vegetative cells
or spores (fewer than 3 cells (0.1–2.5 cells) per gram) (Kaneko et al.,
2011) (Suppl. Table S1). Finally, a combination of a selective enrich­
ment culture together with multiplex PCR has been employed to quan­
tify the different types of C. botulinum spores such as proteolytic spores
which needs a higher temperature to form neurotoxin as well as
non-proteolytic spores which can form neurotoxins at cold temperature
showing high specificity and lower detection limits (Lynt, Kautter, &
Solomon, 1982; Malakar et al., 2013; Peck et al., 2010) (Suppl.
Table S1).
LFIA: Lateral-flow immunochromatographic assay, ELISA: Enzyme-
linked immunosorbent assay, CARD-FISH: Catalyzed reporter deposi­
tion- Fluorescence in situ hybridization, PCR: Polymerase chain reac­
tion, UHT: ultrahigh-temperature, N/A: Not available, BTAs: Bio threat
agents.
3. Conclusion
Bacterial and fungal spores pose a threat to the economy, human and
animal health on many levels, starting with crop spoilage to life-
threatening foodborne diseases. Although many procedures are fol­
lowed for microbial growth inhibition, yet spores can survive many of
them. Sensitive, rapid, and on-site detection methods of spores are a
challenge especially in the food industry due to several risk factors for
cross-contamination i.e., raw materials, personnel, production line. Few
methods were reported to detect spores on-site without prior germina­
tion and very high sensitivity to include surface-enhanced Raman
spectroscopy (SERS) and paper-based immunosensor. Microscopy and
chromatography methods did not show good screening feasibility
especially for the on-site or large-scale monitoring, however coupling
both methods were found more convenient. SERS showed the highest
sensitivity and reproducibility within spectroscopic methods, but with
very limited applications to the different food matrices. More future
research directed towards Raman microscopy applications can provide
better insight on individual cell’s behavior and physiological changes
while vegetation conversion to provide more targets for spore detection.
Compared to bacterial spores, much less is known regarding fungal
spores composition. Biosensors provided the most convenient, rapid, on-
site method without the need for skilled personnel, especially if coupled
with automated data acquisition. It showed promising applications in
different food types and matrices with sensitivity resembling those
attained using molecular tools such as PCR. Several molecular methods
have supported the field to detect bacterial and fungal spores in food and
quantifying their numbers as well. Conventional PCR showed high
sensitivity in detecting spores in both bacteria and fungi, but it lacks the
quantification advantage associated with qRT PCR. For this reason, qRT
PCR became more applicable since it can detect the number of spores in
the food industry such in UHT whole milk. In addition to the previous
methods, the combining of two molecular methods or more, has allowed
rapid, sensitive, and specific detection of spores in food industry. It is
always recommended to choose the right detective method to detect
spores at an early stage before reaching the germination stage.
With regards to spore’s inactivation, applying different inactivation
methods either separately or in combination could lead to an acceptable
reduction level, though to vary depending on the spore-forming
microorganism, initial load, type, contact time, the mechanism of
M.A. Farag et al.
inactivation, and finally the blend of the parameters applied and the
matrix in which spore is found. Considering that each inactivation
method has its advantages and/or limitations, hurdle technology
considered an optimum methodology to develop a suitable inactivation
strategy with maximum efficiency. Further, optimization within each
method ought to be considered i.e., by the addition or removal of several
parameters and modifying their magnitude such as decreasing time of
heat exposure, lowering temperature for thermolabile products,
adjusting water activity, or other different approaches. Several inacti­
vation method combinations should be examined to develop new stra­
tegies for spore’s eradication, maximize the positive outcomes and
minimize the negative drawbacks. Factors such as, green strategy,
safety, efficiency, energy consumption, product quality, effect of by-
products produced, resistance of spores, simplicity, and type of in­
dustry, commercial availability should all be put together when
designing new technologies for spores’ inactivation.
Most of the studies reported in this review assessed the influence of a
single or two variables on the spore’s inactivation while the interaction
between the variables have not been fully examined. Therefore, in order
to optimize the spore’s inactivation conditions, it would be necessary to
simultaneously investigate the influence of different variables on spores
count using statistical designs i.e., response surface methodology (RSM).
Authors contribution
MAF organized the review theme and supervised the writing,
Screening and extraction were done by all authors. Data collection,
interpretation, tables, and figures were done by all authors equally. All
authors contributed to the manuscript writing and approved the final
version.
Declaration of competing interest
Authors declare no conflict of interest whatsoever in this paper. All
authors have read and approved this submission.
Acknowledgment
Dr. Mohamed Farag acknowledges the funding received from the
Alexander von Humboldt foundation, Germany.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.tifs.2020.10.037.
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