Critical Reviews in Food Science and Nutrition

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Recent advances in understanding the control

of disinfectant-resistant biofilms by hurdle
technology in the food industry

Lei Yuan , Faizan A. Sadiq , Ni Wang , Zhenquan Yang & Guoqing He

To cite this article: Lei Yuan , Faizan A. Sadiq , Ni Wang , Zhenquan Yang & Guoqing He
(2020): Recent advances in understanding the control of disinfectant-resistant biofilms by
hurdle technology in the food industry, Critical Reviews in Food Science and Nutrition, DOI:
10.1080/10408398.2020.1809345

To link to this article: https://doi.org/10.1080/10408398.2020.1809345

Published online: 25 Aug 2020.

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CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION
https://doi.org/10.1080/10408398.2020.1809345

REVIEW

Recent advances in understanding the control of disinfectant-resistant biofilms

by hurdle technology in the food industry
Lei Yuana , Faizan A. Sadiqc, Ni Wangb, Zhenquan Yanga, and Guoqing Heb
a
College of Food Science and Engineering, Yangzhou University, Yangzhou, China; bCollege of Biosystems Engineering and Food Science,
Zhejiang University, Hangzhou, China; cSchool of Food Science and Technology, Jiangnan University, Wuxi, China

ABSTRACT KEYWORDS
Modern food processing environment provides an ideal condition for biofilms formation by food- Biofilm; chemical
borne and spoilage microorganisms on different food contact surfaces. It is widely acknowledged disinfection; resistance;
that biofilm has become a serious problem in the food industry, as the biofilm growth mode indu- hurdle technology
ces microbial resistance to chemical disinfection. The persistence of biofilms after cleaning and dis-
infection procedures may result in foodborne illness and food spoilage, emphasizing the
importance of preventing biofilms in food production facilities. The use of conventional disinfec-
tion technologies alone may not help to achieve the goal of producing safe food products with
high quality. Hurdle technology provides a great option for the effective control of biofilms
formed on food contact surfaces. Thus, a better understanding of biofilm behavior in response to
different disinfectants, as well as seeking potential hurdle technologies to control biofilms are
essential. In this review, we discuss the factors that influence the efficiency of disinfectants, and
elaborate possible mechanisms which are behind the apparent high antimicrobial resistance of
biofilms, and as well as mechanisms which are involved in effective hurdle technologies to con-
trol biofilms.

Introduction
A biofilm is defined as a complex assemblage of microorgan-
isms growing on a biotic or abiotic surface and forming spa-
tially organized ecosystems within a self-produced matrix
composed of extracellular polymeric substances (EPS) (Donlan
2002). In the food industry, food processing lines provide an
ideal environment for the formation of biofilms on various
food contact surfaces, mainly due to the complexity of proc-
essing facilities, lengthy production cycles, vast areas for
microbial growth, and foremost, the availability of nutrients
(Lindsay and von Holy 2006). Biofilms formed on food con-
tact surfaces act as a persistent source of microbial contamin-
ation due to their bio-transfer potential, which is a threat to
the microbiological quality and safety of food products, in
addition to the metal corrosion and equipment damage caused
by chemical and biological reactions catalyzed in biofilms
(Brooks and Flint 2008). Therefore, efficient cleaning and dis-
infection practices to remove food-processing related biofilms
are urgently needed in the food industry.
In the global food industry, chemical disinfectants are fre-
quently used with an aim to inactivate microorganisms on
surfaces in order to prevent microbiological contamination of
raw materials and food products. However, bacterial cells
within the biofilm matrix are resistant to chemical disinfec-
tants (10–1000 times), compared to their corresponding
planktonic cells (Acker, Dijck, and Coenye 2014). The phe-
nomenon of enormous resistance toward disinfectants by bio-
film-based bacterial cells has been widely reported in a wide
range of food sectors, including dairy processing (Ziech et al.
2016), bakery (Fink et al. 2017), fresh produce (Bang et al.
2017), fish processing (Papaioannou et al. 2018), and meat
processing (Wang et al. 2018). The microbiota found in food
processing plant surfaces after disinfection procedures is com-
monly reported to be diverse and includes pathogenic and
spoilage bacteria, making removing them from food contact
surfaces a huge challenge (Maes et al. 2019). Moreover,
inappropriate use of chemical agents may result in the spread
of disinfectant-resistant bacteria, as well as the emergence of
cross-resistance to antibiotics (Techaruvichit et al. 2016). In
recent years, an increasing number of studies have been con-
ducted to find an effective way to disinfect food contact sur-
face for the removal of biofilms (Figure 1). Nevertheless, the
available knowledge on biofilm resistance to disinfectants is
still insufficient to improve surface disinfection methods.
Since biofilms represent serious challenges to the food
industry, a series of techniques, including cleaning and dis-
infection, bacteriophages, anti-biofilm enzymes, natural
products, lactic acid bacteria and their bacteriocins, the
modification of food contact surfaces, quorum sensing
inhibitors, and physical treatment, have been widely used to
minimize the accumulation of biofilms in food settings

CONTACT Zhenquan Yang yangzq@yzu.edu.cn College of Food Science and Engineering, Yangzhou University, 196 Huayang West Road, Yangzhou,
Jiangsu 225127, China; Guoqing He gqhe@zju.edu.cn College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Road,
Hangzhou 310058, China.
ß 2020 Taylor & Francis Group, LLC
2 L. YUAN ET AL.
Figure 1. Publications of biofilm control in the food industry (source: web
of science).
(Yuan, Hansen, et al. 2020). However, the single use of these
methods cannot solve the problem of the complete undesir-
able biofilms. Hurdle technology, the combination of two or
more control methods, is a promising technology to effect-
ively remove biofilm cells from food processing facilities, as
they would attack microorganisms in different ways (Khan
et al. 2017). The synergistic reduction in bacterial contamin-
ation on food contact surfaces by hurdle technology has
been successfully proved by many studies (Ban and Kang
2016; Lim et al. 2019; Jung, Park, and Ha 2018; Hussain
et al. 2019; Kim, Lim, and Kim 2019).
In the context of the above discussion, the objective of
this review is to provide an overview of the current know-
ledge related to the factors that influence the efficiency of
disinfectants against biofilms. In addition, we discuss mecha-
nisms which play an important role in conferring biofilms
resistance to disinfectants, as well as the promising hurdle
technologies to control biofilms.
Factors affecting biofilm removal by the single use
of chemical disinfectants
Till now, a large variety of commercialized disinfectants,
including chlorine-based compounds, hydrogen peroxide, per-
acetic acid, quaternary ammonium compounds, and ozone,
have been widely used for reducing the microbial contamin-
ation for the production of safer and longer shelf-life products
(Figure 2). However, considerable studies have already shown
that the single use of chemical disinfection fails to completely
remove biofilm cells on food contact surface, due to resistance
of biofilm cells toward disinfectants s (Table 1). In addition,
many factors including the type and concentration of disinfec-
tants, exposure time, targeted microorganisms, types of surfaces
on which disinfectants are applied, pH, food residues, tempera-
ture, and relative humidity, could affect the efficiency of disin-
fectants to eradicate biofilms (Cappitelli, Polo, and Villa 2014).
Types of disinfectants
Ineffective action of a disinfectant against biofilms may be a
result of the use of an irrelevant or unrecommended
disinfectant, which may have a different mode of action
than the one actually needed. Many studies have compared
the efficiency of different disinfectants. For example,
I~
niguez-Moreno et al. (2018) proved peracetic acid as the
most effective disinfectant to remove biofilms formed by
Staphylococcus aureus and Salmonella spp. on stainless steel
and polypropylene B surfaces, followed by sodium hypo-
chlorite and cetrimonium bromide. Vazquez-Sanchez et al.
(2014) proved that peracetic acid was found to be most
effective against both biofilms and planktonic cells of S. aur-
eus, followed by sodium hypochlorite and benzalkonium
chloride. In addition, Gazula et al. (2019) compared the effi-
cacy of four different sanitizers commonly used by blueberry
packers in removing biofilms, and recommended ozonated
water as a highly efficient sanitizer in removing biofilms,
followed by quaternary ammonium compound, chlorine
dioxide, and sodium hypochlorite.
Disinfectants concentration and exposure time
Reduction of biofilm cells by disinfection is a concentration-
and exposure time-dependent process. In general, disinfection
efficacy against biofilm cells increases with the increasing
disinfectant concentration and contact time. Jung, Park, and
Ha (2018) reported a significant increase in reductions of
Salmonella Typhimurium biofilms on quail eggshell with an
increase in the concentration of sodium hypochlorite. After
treating quail eggshell biofilms with 50, 100, 150, 200, and
300 ppm sodium hypochlorite, reduction of Salmonella
Typhimurium biofilms was 0.1, 0.5, 0.6, 1.9, and 2.2 log CFU/
egg, respectively. Similarly, Wang et al. (2018) reported a sig-
nificant decrease in numbers of surviving biofilm cells follow-
ing disinfection with an increase in exposure time and
disinfectant concentration. More than 3 log reduction of bio-
film cells was observed when chlorine dioxide treatment was
increased from 20 to 40 mg/L.
Types of microorganisms
Disinfectants bind to specific target sites of microorganisms
including outer cellular components, cytoplasmic membrane,
and cytoplasmic constituents. Thus, the efficacy of a disinfec-
tion process depends on different types of microorganisms (or
even different strains of the same species) which are subjected
to a disinfection treatment. For example, Lim et al. (2017)
proved that pathogenic bacteria from a cafeteria kitchen
exhibited a wide range of susceptibility to the disinfection pro-
cess, and the susceptibilities to different disinfectants depends
on different strains. Bayoumi et al. (2012) validated the regular
sanitizing process for the elimination of pathogenic bacteria
isolated from dairy products, and results showed that the effi-
ciency of sodium hypochlorite to remove biofilm-embedded
bacteria depends on bacterial strains.
In addition, varied genetic characteristics of different bac-
terial strains may also be responsible for their varied
response to different disinfectants. EPS acts as a defense bar-
rier that difficult the penetration of disinfectants and help
biofilm cells to resist the disinfection process. Generally,

Figure 2. Overview on the advantages and disadvantages of each disinfectant applied in the food industry.
EPS quantity and composition vary from one bacterial spe-
cies to another, which could affect the efficacy of disinfec-
tants. For example, Enterobacter cloacae biofilms, compared
to Klebsiella oxytoca and Citrobacter freundii biofilms, were
more resistant to sodium hypochlorite, chlorine dioxide,
strongly acidic electrolyzed water, and neutral electrolyzed
water, due to thicker biofilm (Cai et al. 2018). Staphylococci
strains containing the ica operon responsible for production
of a polysaccharide matrix were more resistant to the lethal
effect of benzalkonium chloride than strains without the ica
operon (Fagerlund et al. 2016). In the same study, strains
with qac genes encoding benzalkonium chloride efflux
pumps, could grow at higher concentrations of benzalko-
nium chloride than strains without these genes. In another
study, biofilm tolerance of commensal strains of Clostridium
perfringens to hydrogen peroxide was due to the presence of
oxidative stress enzymes in the biofilm matrix (Charlebois
et al. 2017).
Type of surfaces
Behavior of sessile microorganisms to disinfectants is
strongly influenced by the type of surfaces, because many
surface-related factors such as structure, topography, rough-
ness, and electric properties, affect the efficiency of disinfec-
tants. Schlisselberg and Yaron (2013) provided insights into
possible factors related to metal surfaces (mechanically pol-
ished and brushed, bright annealed, electro-polished, and
untreated coupons) that affect the biofilm susceptibility to
chlorine. Disinfection with chlorine proved to be up to 130
times more effective on electro-polished stainless steel than
on stainless steel surface polished by bright alum, mechanic-
ally sanded or untreated. This study also suggested that sur-
face finish is an important factor when assessing the role of
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 3
surface characteristics in disinfection. It was suggested that
an electro-polished surface is a good choice to make clean-
ing and disinfection programs effective. The use of mechan-
ically-sanded surface was found appropriate in programs
where cleaning and disinfection are not regularly required.
Bang et al. (2014) observed that stainless steel surface is easy
to disinfect followed by glass, plastic, and wood. Higher
resistance of E. coli O157:H7 against sodium hypochlorite
and chlorine dioxide on wooden surfaces was attributed to
the limited penetration of disinfectants into the crevices and
pores on the wooden surface, resulting in protection of E.
coli O157:H7 from disinfectants. In addition, 1-min treat-
ment of different surfaces (stainless steel, low-density poly-
ethylene, polyvinyl chloride, polyester, and rubber) with
chlorine or quaternary ammonium compound showed vari-
ation in disinfection efficacy against L. monocytogenes bio-
films, which shows the important role of topography,
roughness, hydrophobicity, and hydration levels in disinfec-
tion efficacy (Hua et al. 2019). Thus, choosing an optimal
surface that fits to the requirements of the food industry
and does not affect cleaning and disinfection regimes is
highly desirable for hygienic production.
The age of biofilms
The resistance of microorganisms to disinfectants is affected
by the age of biofilms, mainly due to the increased EPS and
biofilm thickness over time. This hypothesis is supported by
the fact that biofilm matrix may hinder the penetration of
effective disinfectant compounds into biofilms. For example,
Nguyen and Yuk (2013) demonstrated that 168-h biofilms
of Salmonella Typhimurium were more resistant to quater-
nary ammonium compounds, peroxyacetic acid and sodium
hypochlorite than 24/48-h biofilms, despite that this effect
4 L. YUAN ET AL.

Table 1. A summary of selected studies investigating the disinfection-resistant biofilms in the food industry.
Disinfection method Food contact surfaces Results Reference
Benzalkonium chloride
1.0 mM, for 30 min 96-well polystyrene microtiter plates Reduced Pseudomonas aeruginosa Machado et al. (2012)
and Escherichia coli biofilm cells by
3 and 1 log after 6 days
incubation of a dual-species
biofilm model.
50 ppm, 18
C for 6 min stainless steel coupons Reduced Listeria monocytogenes and Giaouris et al. (2013)
Pseudomonas putida biofilm cells
by 1.82 and 0.79 log CFU/cm2
after 10 days incubation of a dual-
species model.
50 ppm, 20
C for 6 min stainless steel coupons Reduced Salmonella Typhimurium Gkana et al. (2017)
and Staphylococcus aureus biofilm
cells by 3.00 and 2.43 log CFU/cm2
in a dual-species biofilm model.
100 ppm, for 6 min stainless steel coupons Reduced Serratia liquefaciens and Liu et al. (2017)
Shewanella putrefaciens biofilm
cells by 2 and 3 log CFU/cm2 in a
dual-species biofilm model.
Chlorine dioxide
50 ppm, 30
C for 20 min stainless steel coupons Reduced Escherichia coli biofilm cells Meireles et al. (2017)
by 3.20 log CFU/cm2.
2.5 and 5 ppm, 22
C for 1 min polystyrene surfaces Reduced the 2-day-old biofilm cells Korany et al. (2018)
of Listeria monocytogenes by 2.4-
3.8 log.
20 mg/L, 30 min, pH 7.2-7.3 stainless steel coupon Reduced biofilm cells of Enterobacter Cai et al. (2018)
cloacae, Klebsiella oxytoca, and
Citrobacter freundii by 4.74, 6.42
and 6.41 log CFU/cm2,
respectively.
20 mg/L, 20
C for 30 min stainless steel coupon Less than 3 log CFU/cm2 reduction of Wang et al. (2018)
Pseudomonas fluorescens biofilm
cells were obtained.
Ethanol
70%, for 5 min 96-well microtiter plates Reduced Staphylococcus aureus and Tote et al. (2010)
Pseudomonas aeruginosa biofilm
cells by 96% and 90%,
respectively.
70%, for 1 min 96-well microtiter plates Reduced Aeromonas hydrophila Jahid and Ha (2014)
biofilm cells by 5 log CFU/g.
40%, for 15 min 96-well microtiter plates Reduced Yersinia enterocolitica biofilm Park et al. (2015)
cells by 88%.
75%, for 6 min stainless steel coupons Reduced Serratia liquefaciens and Liu et al. (2017)
Shewanella putrefaciens biofilm
cells by 2 and 3 log CFU/cm2 in a
dual-species model.
Electrolyzed water
Neutral, 50 ppm, 30
C for 20 min stainless steel coupon Reduced Escherichia coli biofilm cells Meireles et al. (2017)
by 3.26 log CFU/cm2.
Acidic, 40 mg/L, pH 2.70-2.90, stainless steel coupon Reduced biofilm cells of Enterobacter Cai et al. (2018)
for 30 min cloacae, Klebsiella oxytoca, and
Citrobacter freundii by 5.91, 6.42
and 7.03 log CFU/cm2,
respectively.
Slightly acidic, 120 ppm, for 5 min stainless steel, glass, Reduced Listeria innocua biofilms on Jeon, Kwon, and Yoon (2018)
polypropylene, rubber food contact surfaces, and the
highest reduction rate was
achieved on
polypropylene (97.33%).
Hydrogen peroxide
0.47%, for 20 min stainless steel coupons Reduced Pseudomonas aeruginosa Rushdy and Othman (2011)
biofilm cells by 4 log CFU/cm2.
2500 ppm, for 30 min 96-well microtiter plates Reduced biofilm cells of Aeromonas Jahid and Ha (2014)
hydrophila by 3.2 log CFU/g.
2%, for 10 min stainless steel coupon Reduced biofilm cells of Listeria Chen, Zhao, and Doyle (2015a)
monocytogenes, Salmonella
Typhimurium, and Escherichia coli
by 0.3, 0.7 and 1.5 log CFU/
coupon, respectively.
2%, 5 min stainless steel coupon Reduced Staphylococcus aureus and Rios-Castillo, Gonzalez-Rivas, and
Enterococcus hirae biofilm cells by Rodriguez-Jerez (2017)
6.12 and 2.69 log CFU/cm2,
respectively.
Ozone
(continued)
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 5

Table 1. Continued.
Disinfection method
45 ppm, for 1 h
1, 2, and 4 ppm, 22
C for 1 min
0.5 ppm, for 20 min
Peracetic acid
0.5%, pH 2.3, for 180 s
100 mg/L, pH 2.8, 25
C for 10 min
0.2%, pH 1.0, for 20 min
0.075%, 25
C for 20 min
Sodium hypochlorite
50 ppm, pH 6.8, for 1 min
0-200 ppm, for 5 min
50 ppm, 30
C for 20 min
144 ppm, 22
C for 3 min
Food contact surfaces
stainless steel, polypropylene and
polished granite coupons
polystyrene surfaces
stainless steel coupon
96-well microtiter plates
stainless steel coupons
stainless steel coupons
stainless steel coupons
stainless steel coupon
cucumber
stainless steel coupon
stainless steel coupon
Results Reference
Reduced the cell Listeria Nicholas et al. (2013)
monocytogenes attached to
stainless steel, granite and
polypropylene by 3.41, 3.42 and
1.11 log CFU/cm2, respectively.
Reduced the 2-day-old biofilm cells Korany et al. (2018)
of Listeria monocytogenes by 0.9-
4.1 log.
Reduce biofilm cells by 1.61-2.14 log Marino et al. (2018)
CFU/cm2 and 3.26-5.23 log CFU/
cm2 under static and dynamic
conditions, respectively.
Reduced biofilm cells of Listeria Lee et al. (2017)
monocytogenes by 1.8-2.5
log cycles.
Reduced Enterococcus faecium and de Castro et al. (2017)
Enterococcus faecalis biofilm cells
by 1.57 and 3.18 log CFU/cm2,
respectively.
Reduced biofilm cells of Bacillus Fernandes et al. (2018)
cereus by 1.9 log CFU/cm2.
Reduced 1.1 log CFU/cm2 of Oxaran et al. (2018)
Staphylococcus aureus and 2 log
CFU/cm2 of Listeria monocytogenes
in a five-species biofilm model.
Reduced biofilm cells of Salmonella Yang et al. (2016)
Enteritidis by 4.08 (formed at 25
o
C) and 4.43 log CFU/cm2 (formed
at 4 oC) in tryptic soy broth.
Reduced biofilm cells of Cronobacter Bang et al. (2017)
sakazakii by 0.89-1.88 log
CFU/cm2.
Reduced biofilm cells of Escherichia Meireles et al. (2017)
coli by 2.46 log CFU/cm2.
Reduced biofilm cells of Listeria Dhowlaghar et al. (2018)
monocytogenes by 4.8 log CFU/cm2

was shown to growth condition dependent. In addition,
Korany et al. (2018) also proved that the antimicrobial effi-
cacies of four tested disinfectants (ozonated water, chlorine,
chlorine dioxide, quaternary ammonium compounds, and
peroxyacetic acid) against 7-day L. monocytogenes biofilms
on polystyrene surface were much lower than those of 2-day
biofilm. Compared to fresh biofilms, mature biofilms are
normally more resistant to disinfections mainly due to
strong three-dimensional structure comprising of adhered
multiple layers of bacterial cells.
However, Yang et al. (2009) found that this age-
dependent biofilm resistance toward disinfectants can only
be observed on smooth surfaces and not on rough surfaces.
This difference may be explained by the surface properties
of rough and smooth high-density polyethylene materials,
which may cause variations in the rate of biofilm matur-
ation. High porosity of rough surfaces provides a larger sur-
face area for bacterial attachment than smooth surfaces,
therefore, biofilm maturation might be faster on rough sur-
faces than on smooth surfaces.
Food residues
The presence of food residues on food contact surfaces
before disinfection significantly influences the cleaning and
disinfection regimes because these residues promote biofilm
formation and also alter the surface properties (Møretrø
et al. 2012). In addition, the presence of organic matter may
also reduce the antimicrobial property of disinfectants. For
example, the soiling of surfaces with chicken juice decreased
the efficiency of sodium hypochlorite against Salmonella and
L. monocytogenes biofilms, because chlorine is rapidly
reduced when it comes in contact with organic matter in
chicken juice (Sarjit and Dykes 2017; Pang et al. 2019).
Small sediments of protein- and carbohydrate-rich food resi-
dues, including milk and egg yolk, reportedly increased the
resistance of surface-adherent spoilage yeasts (Saccharomyces
cerevisiae and Debaryomyces hansenii) and lactic acid bac-
teria to disinfectants, and promoted cross contamination
and food spoilage (Shikano et al. 2017; Kuda, Nakano, et al.
2016). Vegetable sediments, though poor in proteins and lip-
ids, contain polysaccharides that have high water holding
capacity, and thus they might protect the bacterial cells from
disinfectants. In addition, vegetables containing some anti-
oxidants (e.g. ascorbic acid and carotenoids) may also pro-
tect the biofilm cells from the reactive oxygens. Kuda,
Koyanagi, et al. (2016) proved the protective role of carrot
and green bell pepper juice for biofilm cells of Salmonella
Thyphimurium against disinfection treatments by hypo-
chlorous acid, ethanol, and benzalkonium chloride. Thus,
these results suggest that proper and regular washing to
remove all traces of food debris from surfaces allows direct
6 L. YUAN ET AL.
contact of disinfectant and target bacteria, which is essential
for the efficacy of disinfectants.
Other environmental factors
Other environmental factors, including pH, temperature,
and humidity, may also influence the disinfection efficiency.
Many disinfectants have their optimum pH range for their
optimum activity. For example, quaternary ammonium com-
pounds were more effective against L. monocytogenes bio-
films at the higher pH values of 10.42–11.46 as compared to
their efficiency at the pH ranging from pH 6.24 to 8.70
(Yang et al. 2009). Song et al. (2014) found that the pH-
adjusted (pH 7) chlorine solutions were more effective in
reducing bacterial numbers than the pH-unadjusted (pH
8–9) chlorine solutions at the same concentrations. In gen-
eral, increase in temperature of the treatment may increase
the efficiency of disinfectant agents. When disinfection
occurs at low temperatures, the use of higher concentrations
of biocides or elongation of the contact time may increase
effectiveness (Møretrø et al. 2012). Abdallah et al. (2015)
showed that the effect of growth temperature on biofilm
resistance was dependent on the active agents in each disin-
fectant product. The efficiency of disinfection is also affected
by relative humidity. Bae, Baek, and Lee (2012) found that
levels of S. aureus and C. sakazakii biofilms were more
slowly reduced after storage at relative humidity of 23%,
43%, 68%, and 85% for 5 days than the levels of many other
pathogens. Formation of biofilms stored at relative humidity
of 100% for 5 days displayed the highest levels of resistance
to inactivation.
Figure 3. The mechanisms involved in biofilm resistance to chemical disinfectants.
The mechanisms involved in biofilm resistance to
disinfectants
The high resistance of biofilm cells to disinfectants may
increase the risk of disinfection failure, leading to severe
health problems and economic losses. Considerable studies
have concluded that the biofilm resistance to disinfectants is
a multifactorial process resulting from different mechanisms
(Figure 3): (a) reduced penetration of disinfectants into the
biofilm, (b) an altered physiology of the biofilm cells, (c)
protection in mixed-species biofilms, and (d) the occurrence
of persister cells.
Reduced penetration of disinfectants into biofilms
To inactivate microorganisms, disinfectants must achieve a
satisfactory concentration at the target site to perform their
antimicrobial actions. However, it is generally accepted that
the inherent three-dimensional network of cells entangled in
biofilms and EPS, that constitute a compact structure, which
may hinder the penetration of disinfectants into deeper
layers of biofilms, and thereby facilitating the establishment
of ecological niches within the biofilm and protecting the
cells against the actions of antimicrobial compounds
(Flemming and Wingender 2010). For example, the involve-
ment of biofilm matrix in the apparent resistance of biofilms
cells to disinfectants was reported by Jang et al. (2006), who
found the depletion of disinfectant at 100 lm in the biofilm
depth, indicating that the penetration of chlorine dioxide
was delayed in the mixed biofilm of undefined bacteria from
unpasteurized whole milk. The extracellular matrix protein
CabA contributes to the formation of filaments in the

biofilm matrix where the filaments connect bacterial cells
together to build robust biofilms that are resistant to the dis-
infection by sodium hypochlorite (Park, Lee, et al. 2016).
Moreover, Bridier et al. (2012) suggested that the high
resistance of Bacillus subtilis biofilms to peracetic acid was
related to the specific three-dimensional structure of the bio-
film, as a large quantity of extracellular matrix could hinder
the penetration of peracetic acid.
Nevertheless, the diffusion barrier can partly explain the
reduced susceptibility of biofilms to disinfectants. The inter-
actions between disinfectants and the biofilm matrix may
also be responsible for failed penetration of antimicrobials
to deeper layers of biofilms (Davison, Pitts, and Stewart
2010). The biofilm matrix may involve either electrostatic or
hydrophobic interactions in order to hinder the penetration
of antimicrobial agents into deeper layers biofilms (Zhang,
Nadezhina, and Wilkinson 2011). For example, using time-
lapse confocal laser imaging, Davison, Pitts, and Stewart
(2010) found that the low penetration of quaternary ammo-
nium compound to the center of Staphylococcus epidermidis
biofilm cluster takes 60-times longer than the time required
for diffusive movement in the absence of sorption the posi-
tively charged and hydrophobic disinfectant. In another
study, a biofilm matrix dominated by polysaccharides pro-
tected staphylococci against benzalkonium chloride in a bet-
ter way than a matrix dominated by proteins. One possible
explanation may be the reduced diffusion of the positively
charged benzalkonium chloride in a biofilm in which nega-
tively charged polysaccharide intercellular adhesin is a major
matrix component (Fagerlund et al. 2016). This study also
suggested that the deactivation of reactive compounds in
disinfectants is affected by the presence of organic matters
of the biofilm matrix (e.g. polysaccharides, proteins, and
nucleic acids). Using time-lapse confocal laser imaging com-
bined with Chemchrome V6/Chemsol B16 staining, Bridier
et al. (2011) observed that the matrix of P. aeruginosa
delayed the penetration of benzalkonium chloride, which
was probably because of the interactions between biofilm
components and the antimicrobial. In addition, the presence
of degradative enzymes such as catalases in the biofilm
matrix of P. aeruginosa has also been reported to prevent
the penetration of hydrogen peroxide into biofilms, whereas
catalase-deficient biofilms have been reported to be suscep-
tible to hydrogen peroxide (Stewart et al. 2000).
An altered physiology of biofilm cells
Previous studies have demonstrated the differences between
planktonic and attached bacteria that imply physiological
alterations following attachment to a surface. This “biofilm
phenotype” was proposed to explain the resistance of biofilm
cells to disinfections. For example, the up-regulation of
genes involved in EPS production of surface-attached bac-
teria could increase the resistance to disinfections (Friedman
and Kolter 2004). The bacterial membranes, composing of
phospholipids and proteins, serve as the first line of bacterial
defense against disinfectants. The transition to the sessile
phenotype can induce changes in the fatty acid profile of
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 7
bacterial membranes, which maintain the membrane fluidity
of cells (Gianotti et al. 2008). Such an increase in membrane
rigidity may prevent disinfectants penetrating into the lipid
bilayers and enhance the resistance of biofilm cells to disin-
fectants agents at the cellular level (Bisbiroulas et al. 2011;
Abdallah et al. 2014). Another possible mechanism of bio-
cide resistance is based on the observation that some of bio-
film cells are able to sense the biocide challenge and actively
respond to it by deploying protective stress responses more
effectively than planktonic cells. For example, Sanderson
and Stewart (1997) reported that the second dose of mono-
chloramine, when given to P. aeruginosa biofilms, was less
effective than the first dose, and similarly each subsequent
dose proved to be less effective. P. aeruginosa biofilms also
showed increased catalase (katB) expression during treat-
ment with hydrogen peroxide at a concentration sublethal to
biofilm cells. Bacterial growth in the deeper layer of biofilm
is reduced owing to nutrient and oxygen limitation, and it
may diminish the effectivity of antimicrobials, which is also
recognized as means of survival for bacteria in biofilms that
are exposed to disinfectants (Flemming et al. 2016).
Furthermore, the up-regulation or induction of genes coding
multidrug efflux pumps in biofilms may be another possible
mechanism to explain resistance to antimicrobials in bio-
films (Mah and O’Toole 2001). Efflux pumps are systems
that enable cells to release toxic molecules out of their cells
and allow bacteria to survive in the presence of these sub-
stances. One example of a well-known system specific to
biocides is the quaternary ammonium compounds efflux
system of S. aureus which is responsible for its high level of
resistance to quaternary ammonium compounds and cat-
ionic biocides (Smith and Hunter 2008).
Protection in mixed-species biofilms
In the food processing environment, surface-associated bacter-
ial communities are usually complex associations of different
bacterial species, which interact in different ways to constitute
a complex and dynamic network. Such interactions play a key
role in shaping biofilm architecture and are responsible for
specific functions including the resistance to disinfectants
(Yuan, Hansen, et al. 2020). Generally, mixed-species biofilms
are less susceptible to disinfectants when compared to their
corresponding mono-species biofilms (Yuan, Hansen, et al.
2020). For example, Salmonella Enteritidis in a dual-species
biofilm with P. aeruginosa was less susceptible toward sodium
hypochlorite, with the 1 log CFU/cm2 lower reduction than
that in mono-species biofilms, despite the observation that the
presence of P. aeruginosa reduces the population of
Salmonella Enteritidis (Pang and Yuk 2018). The dual-species
biofilm formed by Pseudomonas libanensis and Aeromonas
hydrophila was more tolerant of hydrogen peroxide, peracetic
acid, and sodium hypochlorite when compared to each single-
species biofilm (Yuan, Wang, et al. 2020). In addition,
Schwering et al. (2013) found that multi-species biofilm com-
munities comprising of natural microflora were able to survive
chlorine concentrations up to 375 times the maximum
8 L. YUAN ET AL.
legislated limit, and were 84-fold more resistant than the aver-
age biofilm tolerance of the corresponding single-spe-
cies biofilm.
The possible mechanisms of enhanced resistance to disin-
fectants within mixed-species biofilms are: (a) changed com-
position of the matrix and enhanced EPS production in
mixed-species biofilms; (b) the specific spatial arrangement
of certain bacterial species within a biofilm, as some strains
may be protected from a biocide by their aggregation with
others within the differential three-dimensional structure; (c)
transient changes in proximal neighbors, as one species
residing within a mixed-species biofilm can significantly
alter the physiology and thus enhance the resistance of
neighboring species by interspecies interactions (Yuan,
Hansen, et al. 2020).
Nevertheless, it should be noted that mixed-species bio-
films are not always more resistant to disinfectants, as the
susceptibility to disinfectants can be attributed to factors
including types of food contact surfaces and disinfectants,
experimental methods used, and the type of included micro-
organisms as well as their specific interactions (Yuan,
Hansen, et al. 2020).
Persister cells
Persister cells describe a bacterial phenotype of bacterial cells
in biofilms, in which a small fraction of bacterial cells (usu-
ally 0.1–10% of the total biofilm cells) show antimicrobial-
resistance. Recently, the involvement of persister cells has
been proposed as another explanation for the reduced sus-
ceptibility of biofilms to disinfectants, as the overuse of dis-
infectants may create selective pressure favoring emergence
of adaptive and cross resistances and their transfer to nonre-
sistant microflora (Lewis 2010). Such isolates in food proc-
essing scenarios may render routine cleaning and
disinfecting regime less effective. Sim~ oes et al. (2011) indi-
cated the possibility of the existence of persister cells among
P. fluorescens biofilm cells and the extreme capacity of bio-
film cells to recover from exposure to ortho-phthalaldehyde
treatment, even without the protection of EPS.
Combining disinfection strategies –
hurdle technology
The new concept of “hurdle technology” provokes the intel-
ligent use combinations of physical–chemical, chemical–-
chemical, or biological–chemical disinfection methods to
effectively control undesirable microbial biofilms by hitting
different targets within the bacterial cells at the same time
(Table 2). In the food industry, disinfection should be per-
formed cost-effectively and safely, which means it should be
performed less frequently, if possible, and within the short-
est possible time, with low chemical, energy and labor costs,
producing as little waste as possible and with no damage to
the equipment. In the food industry, hurdle technology is
expected to have greater effectiveness at controlling biofilms
than the single use of chemical disinfectants (Figure 4).
Thus, the potential solutions for the combined disinfection
procedures should be carefully chosen to achieve an
adequate disinfection effect.
Physical–chemical disinfection
Many studies have evaluated the inactivation efficiency of
the combined use of physical and chemical disinfection
methods, compared with that of individual treatments. Food
irradiation has the ability to damage microbial DNA without
having any negative effects on the sensorial or nutritional
quality of the food, when an appropriate irradiation dose is
applied (Farkas and Mohacsi-Farkas 2011). X-ray irradiation,
which has a deeper penetration potential than gamma rays,
has a considerable advantage, specifically when using an on
and off system with a switch (Farkas and Mohacsi-Farkas
2011). The synergistic reduction of Salmonella enterica sero-
var Typhimurium biofilm cells on eggshells was 4.3 log
CFU/egg after the combined treatment by 2.0 kGy X-ray
and 50 ppm sodium hypochlorite, with 1.47 log higher than
the sum of reduction values of the individual treatment, and
biofilms on eggshells did not exhibit a three-dimensional
structure (Jung, Park, and Ha 2018). Moreover, this combin-
ation did not cause any color or thickness changes of quail
eggshell. Additionally, ultraviolet irradiation has many
advantages including no requirement of heat or chemicals,
relatively low cost and less effect on the nutritional quality
of foods (Kim, Park, and Ha 2016). Previous studies have
demonstrated that the combined use of ultraviolet irradi-
ation and chemical disinfection are highly effective against
biofilm cells. The synergistic effect of a combined treatment
of biofilms with hydrogen peroxide (5 ppm) and ultraviolet
irradiation (234 mJ/cm2) was shown to be much more effect-
ive than the single use of hydrogen peroxide by 10-fold,
which could contribute to a more environmental-friendly
treatment (Vankerckhoven et al. 2011). In another study,
synergistic reductions of L. monocytogenes biofilm cells on
stainless steel and eggshells after the treatment by ultraviolet
irradiation/sodium hypochlorite were observed, and the larg-
est synergistic reductions were 3.68 log CFU/cm2 and 1.02
CFU/cm2 on stainless steel and eggshells when using 1800
mWs/cm2 ultraviolet irradiation and 200 ppm sodium hypo-
chlorite or 600 mWs/cm2 ultraviolet irradiation and 50 ppm
sodium hypochlorite, respectively (Kim, Park, and Ha 2016).
The explanation for this synergistic effect is the combined
bacterial inactivation mechanisms: ultraviolet irradiation
damages DNA and RNA of bacteria and sodium hypochlor-
ite destroys bacterial cell wall.
The ultrasound treatment is a nonchemical and environ-
mentally friendly technology to disrupt biofilm structure,
which releases cells in their planktonic state or even inacti-
vates microorganisms (Yu et al. 2020). Ultrasound treatment
is more effective when used in combination with chemical
disinfection, as the cells in the deeper layers of mature bio-
films are stimulated by the ultrasound that makes them
more susceptible to damage by chemical disinfectants. For
example, synergistic reduction of C. sakazakii biofilms was
observed in a combined treatment of ultrasound and per-
oxyacetic acid, as cells of C. sakazakii were loosely
 CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 9

aggregated and scattered after the cavitation of ultrasound,
and further destroyed and aggregated in intact and regular
shapes by the peroxyacetic acid treatment. The highest syn-
ergistic reduction in fresh cucumber was 3.51 log CFU/cm2
when treated with a combination of 60 min ultrasound
(37 kHz, 380 W) and 150–200 ppm peroxyacetic acid, which
may help enhance their shelf-life during transportation and
storage without any changes in food quality (Bang et al.
2017). Similarly, the combined treatment of ultrasonication
(37 kHz, 380 W) for 100 min and sodium hypochlorite
(200 ppm) resulted in the highest biofilm cells reduction of
C. sakazakii (4.44 log CFU/g), which was greater than either
with the single treatment with ultrasonication (0.58 log
CFU/g) or sodium hypochlorite (2.77 log CFU/g) (Park,
Mizan, et al. 2016). For the combined treatment of slightly
acidic electrolyzed water (80 mg/L), mild heat (60
C) and
ultrasound (40 kHz, 400 W) for 12 min, additional reductions
of Bacillus cereus ATCC 10987 biofilm cells were achieved
by 1.63, 1.39, and 1.49 log CFU/cm2 on spinach, beet leaf
and lettuce coupons, respectively (Hussain et al. 2019). The
mechanism behind this synergistic reduction is an increase
in the penetration depth of active chlorine compounds of
slightly acidic electrolyzed water through the plasma mem-
brane of the attached B. cereus as a result of the pressure
developed during the sonication with high temperature. In
the bakery industry, Fink et al. (2017) found that the use of
either 30 min ultrasonic treatment (37 kHz, 200 W) or 2%
commercial disinfectant (10% alcohols, 2.5% benzakolium
chloride, and 2.5% didecyldimethylammonium chloride)
alone did not totally remove the biofilms cells. However, the
highest efficacy of B. cereus biofilm removal was achieved
with their combination. In the fruit processing industry, the
combined treatment of ultrasound and chemical disinfec-
tants on cherry tomatoes was performed to achieve a higher
reduction rate of tested microbes compared with the reduc-
tion achieved with the use of a single method (either ultra-
sonication or chemical disinfectants) under the same
conditions. The highest reductions of aerobic mesophiles
(4.4 log CFU/g), molds and yeast (3.4 log CFU/g) on cherry
tomatoes were achieved with a 10 min-combined treatment

Table 2. A summary of recent literature investigating the biofilms control by hurdle technology.
Hurdle technology
Microorganisms Food contact surfaces Reduction log Synergistic valuea References
A B
Escherichia radish seeds chlorine dioxide drying (25 oC, 24 h) 3.8 1.46 Kim et al. (2010)
coli O157:H7 (200 lg/
mL, 5 min)
Salmonella cherry tomatoes peracetic acid ultrasound 3.88 1.09 Jose and
Typhimurium (40 mg/L) (45 kHz, 10 min) Vanetti (2012)
Listeria polyvinyl chloride lactic acid (2%, steam (20 s, 3.99 1.40 Ban et al. (2012)
monocytogenes 30 s) absolute pressure
of 143 kPa)
Escherichia wooden coupons aqueous chlorine drying (43% RH and 6.4 2.78 Bang et al. (2014)
coli O157:H7 dioxide (200 ug/ 22
C for 12 h)
ml, 10 min)
Escherichia spinach leaf organic aid (2% electrostatic 4 1.82 Almasoud
coli O157:H7 malic acid þ 2% spraying (twice et al. (2015)
lactic acid) for 5 s)
Cronobacter head lettuce sodium ultrasound (37 kHz, 4.44 1.67 Park, Mizan,
sakazakii hypochlorite 380 W, 100 min) et al. (2016)
(200 ppm)
Listeria stainless steel sodium ultraviolet-C (1800 6.7 2.22 Kim, Park, and
monocytogenes hypochlorite mWs/cm2) Ha (2016)
(200 ppm)
Escherichia stainless steel sodium hypochlorite steam (20 s, 4.03 1.32 Ban and
coli O157:H7 (20 ppm, 30 s) absolute pressure Kang (2016)
of 143 kPa)
Cronobacter cucumber peroxyacetic acid ultrasound (37 kHz, 3.51 1.42 Bang et al. (2017)
sakazakii (200 ppm, 5 min) 380 W
for 60 min)
Salmonella enterica quail eggshells sodium hypochlorite X-ray irradiation 4.3 1.54 Jung, Park, and
serovar (50 ppm, 3 min) (2.0 kGy) Ha (2018)
Typhimurium
Salmonella stainless steel coupon levulinic acid sodium dodecyl 6.9 11.5 Chen, Zhao, and
Typhimurium (3%, 10min) sulfate Doyle (2015a)
(2%, 10 min)
Escherichia stainless steel coupons phytic acid sodium chloride 6 3.33 Kim and
coli O157:H7 (0.2%, 5 min) (4%, 5 min) Rhee (2016)
Salmonella stainless steel coupons sodium hypochlorite proteinase K (1 3.4 1.36 Kim, Lim, and
Typhimurium (20 ppm, pH 7.4, mAU, 25
C for Kim (2019)
25
C for 1 min) 1 h)
Escherichia stainless steel coupons sodium hypochlorite proteinase K (1%, 37 6.15 2.03 Lim et al. (2019)
o
coli O157:H7 (20 ppm, 10min) C, 1 h)
Salmonella. spp stainless steel coupons cetyltrimethyl cellulase (20 mg/mL, 6.2 0.56 Wang et al. (2016)
ammonium 40 oC, 2 h)
bromide
(1 mg/mL)
aSynergistic value ¼ Log reduction of biofilm cells after the treatment by hurdle technology / (the sum of log reduction after the treatment by each single
method. Synergistic values in Table 2 are derived from the results in the references.
10 L. YUAN ET AL.
Figure 4. Increased reduction of biofilm cells by hurdle technology: (A) disinfection process by the single use of chemical disinfection; (B) disinfection process by
the combination of chemical disinfection and other methods.
of ultrasonication (45 kHz) and 40 mg/L peracetic acid with-
out losing any quality of the product (Jose and Vanetti
2012). All these aforementioned results indicate that chem-
ical disinfectants combined with ultrasound have great
potential as promising approaches for sanitizing food equip-
ment and real food surfaces.
Steam treatment has proven to be a rapid and effective
method for inactivating biofilms in food processing environ-
ments. During steam treatment, the condensation of steam
onto coupon surfaces produces a transfer of heat energy,
which causes rapid heating of the coupon surface, and is
able to effectively penetrate into cavities, crevices, and fea-
ther follicles that may provide protection for surface-
attached bacteria, thus effectively destroying any pathogen
biofilms which are otherwise difficult to eradicate (Ban and
Kang 2016). Hence, steam-based treatments need to be
developed as effective and time-saving approaches for inacti-
vating and detaching foodborne pathogens from food con-
tact surfaces. Steam-based treatments can be used in
combination with aqueous sanitizers, hot water, and ultra-
sound to achieve better results. For instance, there was a
synergistic effect of the combined use of a sanitizer and
steam treatment on the viability of biofilm cells of E. coli
O157:H7, Salmonella Typhimurium and L. monocytogenes.
The combined treatment resulted in an additional 0.01-2.78
log reduction compared to the sum of each individual treat-
ment. The most effective combination for reducing biofilm
cells was the combination of steam (20 s) and iodophor
(20 ppm for 30 s), which reduced cell numbers to below the
detection limit (Ban and Kang 2016). A similar study
showed that the combination of steam (20 s) and lactic acid
(2% for 30 s) reduced biofilm cells of E. coli O157:H7,
Salmonella Typhimurium, and L. monocytogenes to below
the detection limit (Ban et al. 2012).
The combination of aqueous chlorine dioxide (200 lg/ml,
10 min) and drying (43% RH and 22
C for 12 h) also
showed synergistic effects on the removal of biofilms formed
by E. coli O157:H7 on wooden surfaces (Bang et al. 2014).
Similar effects of the combined use of aqueous chlorine
dioxide and drying have been reported for E. coli O157:H7
biofilms on radish seeds (Kim et al. 2010). The possible rea-
son for the enhanced reduction of E. coli O157:H7 biofilms
is the sublethal injury to cells caused by chlorine dioxide,
which makes them more susceptible to additional stresses.
Advanced active-delivery methods, including electrostatic
spray technology have revolutionized industrial and agricul-
ture spraying system. In an electrostatic spray, charged par-
ticulates which are smaller than 100 lm are electrostatically
deposited on a surface. Synergistic reductions of E. coli
O157:H7 on spinach leaf (4.14 log reduction) and
Salmonella Typhimurium (3.60 log reduction) on cantaloupe
rind was achieved when electrostatic spray containing syn-
thetic organic acids (2% malic acid þ 2% lactic acid) was
applied (Almasoud et al. 2015).
Chemical–chemical disinfection
The use of biocidal solutions containing more than one bio-
active compound was also proved to be useful in removing
biofilms on food contact surfaces (Morente et al. 2013). For
instance, biofilm cells of L. monocytogenes were completely
eliminated from stainless steel when treated with a mixture
of disinfectants of quaternary ammonium compounds and
hydrogen peroxide, or peracetic acid, hydrogen peroxide,

and octanoic acid, while single active agent in the disinfectant
failed to completely remove biofilm cells (Dhowlaghar et al.
2018). Rios-Castillo, Gonzalez-Rivas, and Rodriguez-Jerez
(2017) indicated that mixing different disinfectants may facili-
tate their penetration into the biofilm cells or that enhance its
oxidizing action, leading to an acceptable bactericidal efficacy
on stainless steel surfaces even at a reduced concentration of
disinfectants. Aryal and Muriana (2019) proved that the best
performing sanitizer across all three pathogens (L. monocyto-
genes, E. coli O157:H7, and Salmonella serovars) was a com-
bination of modified quaternary ammonium chloride,
hydrogen peroxide, and diacetin, which resulted in 6–7 log
reduction and reached levels below the detection limit within
only 1–2.5 min. Phytic acid is a natural plant compound with
the special structure of 12 replaceable protons that are
responsible for its strong ability to “chelate” positively
charged ions. The combination of phytic acid (0.4%) and
sodium chloride (2–4%) completely inactivated E. coli
O157:H7 biofilms on stainless steel without any recovery,
while neither phytic acid nor sodium chloride alone was
effective (phytic acid, 1.6–2.7 log CFU/cm2 reduction; sodium
chloride, 0.5 log CFU/cm2 reduction) (Kim and Rhee 2016).
The reason for this effect is the damage to bacterial cells in
biofilms caused by phytic acid, which makes the cells vulner-
able to further damage from Naþ and Cl.
Surfactants, like sodium dodecyl sulfate, can act as antiad-
hesive agents and levulinic acid may assist in removal of the
attachment polymer by chelating divalent cations required to
link the polymer to the surface. Hence, the combination of
an organic acid and surfactant may increase the activity of
each other, which is helpful in detaching bacterial cells from
a biofilm matrix. Once the cells are directly exposed to the
chemicals applied without the protective effect of EPS,
sodium dodecyl sulfate can chelate divalent cations, such as
Ca2þ and Mg2þ, which cause instability of outer membrane
of Gram-negative bacteria. Chen, Zhao, and Doyle (2015a)
found that a synergistic effect was observed to inactivate the
biofilms formed by L. monocytogenes, Salmonella
Typhimurium, and Shiga toxin-producing E. coli on stainless
steel coupon, when treated with the combination of levulinic
acid (3%) and sodium dodecyl sulfate (2%) for 10 min.
Similarly, the same combination also led to a synergistic effect
to inactivate the mixed-species biofilms formed by E. coli
O157:H7 (7.1 log CFU/mL) and Salmonella (8.4 log CFU/
mL) (Chen, Zhao, and Doyle 2015b).
Biological-chemical disinfection
In industrial settings, enzymatic treatments have been pro-
posed as an efficient and environmentally-friendly way to
degrade components of the matrix components and facilitate
penetration of cleaning and disinfection agents into biofilm
cells in deeper layers (Nahar et al. 2018). Nevertheless, the
single use of enzymes for the eradication of biofilms in the
food industry generally lack biocidal activity, making them
inappropriate for bactericidal purposes. To overcome this
issue, a combination of enzymatic and chemical approaches
is desirable, since the action of the enzyme would positively
CRITICAL REVIEWS IN FOOD SCIENCE AND NUTRITION 11
contribute to the killing capacity of the disinfectant. This
approach has the advantage of reducing the excessive use of
toxic antimicrobial agents. In addition, the use of disinfec-
tants in combination with protease-based enzymes can be
more helpful in removing biofilm mass residues, which can
prevent the adherence of secondary colonizers after the anti-
microbial treatment (Rodrıguez-L opez et al. 2017). For
example, Lim et al. (2019) found that proteinase K may
cause defects in the biofilm structure and reduce the barrier
properties, thereby facilitating sodium hypochlorite penetra-
tion and reducing the survivability of cells. The increased
sensitivity of biofilm cells to sodium hypochlorite after
exposure to proteinase K led to a great synergistic inactiva-
tion of E. coli O157:H7 biofilms (6.15 log CFU/cm2 reduc-
tion). In another study, two combinations (100 lg/mL
pronase þ 2000 lg/mL benzalkonium chloride, or 1000 lg/
mL pronase þ 2000 lg/mL benzalkonium chloride) reduced
the L. monocytogenes–E. coli dual-species biofilm cells below
the detection limit, and prevented secondary colonizers from
further adhering after the antimicrobial treatment
(Rodrıguez-Lopez et al. 2017). Moreover, the combination of
cellulase (20 mg/mL, 2 h) and cetyltrimethyl ammonium
bromide (1 mg/mL, 1 h) has been reported to completely
remove all of the Salmonella cells in mature biofilms (6.22
log CFU/cm2 reduction) (Wang et al. 2016). Bacteriophages
are viruses that may release polysaccharide depolymerase
enzymes, which bind to the capsular material of bacterial
cells, during infection. Use of these phage enzymes repre-
sents a natural, highly specific, and nontoxic approach of
controlling microorganisms involved in biofilm formation
(Hansen et al. 2019). A heat-stable polysaccharide depoly-
merase that could effectively degrade bacterial exopolysac-
charide was prepared from the phage infecting Klebsiella,
and this phage enzyme showed a rapid decrease in the
amount of biofilm cells. The elimination rate approached
the maximum (80%) after 4 h of treatment. Enzyme pre-
treatment could also increase the disinfection effect of chlor-
ine dioxide. Approximately 92% of the biofilm bacteria were
eliminated after treatment with the phage enzyme followed
by 30 min of treatment with chlorine dioxide (100 mg/mL).
According to the results based on colony counting and
observation taken from scanning electron microscopy, the
phage enzyme could effectively reduce the attachment of
bacteria as well as the adhesion of EPS in the biofilm. This
study has demonstrated that the phage-borne polysaccharide
depolymerase enzyme are useful for the eradication of bac-
terial biofilms (Chai et al. 2014). Nevertheless, the disadvan-
tages of using enzymes are the relative high cost and low
commercial accessibility of different enzymes. In addition,
the activity and efficiency of enzymes are significantly influ-
enced by many complex environmental conditions (e.g. tem-
perature, pH, water hardness, substrate, food residues, and
varieties of food processing surfaces) in the food industry
(Nahar et al. 2018).
Bacteriocins have been given the status of ‘generally
regarded as safe’ (GRAS), and they also play an important
role in preventing initial cell adhesion and biofilm forma-
tion. Previous studies have showed that enterocin could
12 L. YUAN ET AL.
enhance the efficacy of biocides against sessile cells. For
example, the combination of benzalkonium chloride (1.0 g/
L) and enterocin AS-48 have shown to reduce biofilm viable
cells on stainless steel coupons below detectable levels (6.14
log CFU/mL), which was greater than the single use of ben-
zalkonium chloride at the same concentration (4.1 log CFU/
mL) (Burgos et al. 2017). Similarly, synergistic effects
between bacteriocins and disinfectants were also observed by
Caballero G omez et al. (2013), as the addition of enterocin
AS-48 at the concentration of 50 mg/mL enhanced the effi-
cacy of many disinfectants to remove the methicillin-resist-
ant S. aureus biofilm cells below the detection limit. The
mechanism for the synergistic effects is that disinfectants
damages the bacterial cell wall and outer membrane of bac-
teria, facilitating the diffusion of bacteriocin molecules to
the bacterial cytoplasmic membrane, which is the primary
target for enterocin AS-48.
Other options
Stainless steel is an ideal material for fabricating surfaces and
machinery equipment due to its mechanical characteristics,
expansion coefficient, thermal conductivity, and ease of use,
physicochemical stability, and high resistance to corrosion.
Nevertheless, many bacterial species belonging to industrial
and natural settings tend to form biofilms on stainless steel
surface, which remain intact or less affected even after clean-
ing and disinfection. Thus, attention should be paid toward
design of the equipment and physical properties of the mater-
ial being used. In a previous study, the electro-polished finish
makes biofilm cells much more sensitive to the chlorine treat-
ment, and disinfection was mostly effective on the electro-
polished finish surface with only 0.005% of cells surviving the
treatment (Schlisselberg and Yaron 2013).
Essential oils are natural compounds, which have poten-
tial to serve as alternative natural disinfectants suitable for
the control of biofilm. The increased efficacy of disinfectants
by essential oils was reported by Vazquez-Sanchez, Galv~ao,
Mazine, et al. (2018), who reported an increase in the disin-
fection efficiency of peracetic acid against S. aureus biofilm
cells on stainless steel and polystyrene surfaces when com-
bined with Lippia sidoides. In another study by Vazquez-
Sanchez, Galv~ao, Ambrosio, et al. (2018), combinations of L.
sidoides with Thymus vulgaris or peracetic acid showed the
highest efficacy against 24-h-old L. monocytogenes biofilms
on stainless steel and polystyrene surfaces, causing a syner-
gistic lethal effect. The explanation for the synergistic effect
is based on the activity of essential oils affects the integrity
of bacterial cell wall and membrane, causing damage in
membrane proteins and the disruption of the proton motive
force, thereby increasing the penetration of disinfectants
into cells. In addition, Cui, Ma, and Lin (2016) observed a
synergistic effect on the removal of biofilms formed by E.
coli O157:H7 on lettuce as a result of the combined effect of
1 mg/mL clove oil and 400 W cold nitrogen plasma, without
causing any negative effect on the quality of lettuce. They
suggested that the synergetic antibacterial mechanism of this
combination is as a result of the damage to the bacterial cell
wall and the outer membrane, leading to leakage of cellular
components, such as nucleic acid and ATP.
Future research perspective
In the global food industry, the current disinfection process
mainly relies on the application of chemical disinfectants,
and intensive efforts have been dedicated to improving the
efficiency of chemical disinfection to inactivate biofilms
formed on food-contact surfaces. However, the chemical-
based biofilm removal strategies have been shown to facili-
tate the selection and emergence of disinfectant-resistant
microorganisms that can persistently colonize the processing
environment. Extensive studies have been conducted for the
development of effective, economic, and sustainable tools to
inactive biofilm-associated cells from food processing envi-
ronments. Researchers are advised to combine more than
one biofilm control strategies; for instance, the use of chem-
ical disinfectants can be used in combination with many
other chemical and techniques to eradicate biofilms from
food processing environment. Combining different methods
may result in synergistic removal of biofilm cells because as
we discussed in the above sections. However, exploring right
combinations of control methods is very important, as
inappropriate combinations may induce antagonistic effects
and enhance biofilm formation by decreasing anti-biofilm
activity. It is also important to consider if the used combina-
tions are safe to be used on food contact surfaces and they
do not leave any chemical residues on the surface. Before
designing the control strategies, a better understanding of
biofilm formation process, especially mixed-species biofilms,
is necessary from the early stages to the maturation, in
terms of their physical, chemical and biological phenomena.
In addition, in many cases, the efficiency of a disinfection
process is measured after dislodging sessile cells from food
contact surfaces by swabbing, vortexing, or sonication, fol-
lowed by culture-based approach. However, such sampling
methods only enable the partial recovery of adhering cells,
and that the cells sampled may not be representative of the
physiological state of the whole community, as a biofilm
even formed by a single species is a very heterogeneous
community. Therefore, the effectiveness of surface disinfec-
tion is usually overestimated. In this context, we urgent sci-
entists to standardize the methods for the evaluation of
disinfection efficacy on surface-associated cells.
Conclusions
It is widely accepted that biofilm cells in food processing
environments are resistant to standard cleaning and disin-
fection program. This may lead to increased microbial con-
tamination in both the food processing environments and
the subsequent food products, leading to food spoilage and
in many cases food safety issues. Even though up to now,
conventional biofilm control strategies are still used, where
the need of the time is to develop a more economical and
environmental friendly control strategies for the production
of safe and high-quality food products. Hurdle technology is

a novel and promising technique to fight with biofilms,
since it improves the chemical disinfection efficiency,
reduces the amounts of disinfectants used, saves energy, and
duration of the treatment. Now, worldwide researchers are
dedicated to study novel hurdle technologies for face the
challenges of biofilms in the food industry. More fundamen-
tal studies regarding biofilm resistance to disinfectants are
still of crucial importance in order to design effective, envir-
onment friendly, and practical biofilm control hurdle-
concept based strategies in the future. Additionally, we must
acknowledge that product quality is a top priority for food
manufacturers, therefore, biofilm control strategies should
not compromise the taste, and texture of food products in
any way.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by National Natural Science Foundation of
China (Grant No. 31772080).
ORCID
Lei Yuan http://orcid.org/0000-0002-3139-7800
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