Food Control 31 (2013) 572e585

Contents lists available at SciVerse ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Review

Biofilm formation in food industries: A food safety concern

Sokunrotanak Srey, Iqbal Kabir Jahid, Sang-Do Ha*
School of Food Science and Technology, Chung-Ang University, 72-1 Nae-Ri, Daedeok-Myeon, Anseong-si, Gyeonggi-do 456-756, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Foodborne diseases have always been a threat to human health. They are considered an emergent public
Received 6 July 2012 health concern throughout the world. Many outbreaks have been found to be associated with biofilm. It
Received in revised form is well documented that biofilm has become a problem in food industries as it renders its inhabitants
26 November 2012
resistant to antimicrobial agents and cleaning. In this review, biofilm formation in dairy, fish processing,
Accepted 1 December 2012
poultry, meat, and Ready-To-Eat foods industries are discussed, as well as the biofilm forming abilities’ of
various microorganisms and the influence of food contact surface materials on biofilm formation. In
Keywords:
addition, the conventional and emergent control strategies used to gain more proximity to efficiently
Biofilms
Food industry
maintain good hygiene throughout food industries is discussed.
Control strategies Ó 2012 Elsevier Ltd. All rights reserved.
Chemical control
Novel control strategies
Hurdle technology

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .573
2. Biofilm and development steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .573
2.1. Initial attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
2.2. Irreversible attachment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
2.3. Early development of biofilm architecture (microcolony formation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
2.4. Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
2.5. Dispersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
3. Biofilm formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .574
3.1. Biofilm forming strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574
3.2. Food contact surface materials for biofilm formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
4. Biofilm: problems in food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .575
4.1. Produce industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
4.2. Dairy industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
4.3. Fish processing industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
4.4. Poultry industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
4.5. Meat industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
4.6. Ready-to-eat (RTE) foods industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
5. Biofilm control strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .578
5.1. Cleaning and disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
5.2. Clean-in-Place (CIP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
5.3. Chemical-based control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
5.3.1. Sodium hypochlorite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
5.3.2. Hydrogen peroxide (H2O2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
5.3.3. Ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
5.3.4. Peracetic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

* Corresponding author. Tel.: þ82 31 670 4831; fax: þ82 31 765 4853.
E-mail address: sangdoha@cau.ac.kr (S.-D. Ha).

0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2012.12.001
 S. Srey et al. / Food Control 31 (2013) 572e585 573

5.4. Other approaches of biofilm control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580

5.4.1. Ultrasonication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
5.4.2. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
5.4.3. Phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
5.5. Hurdle technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581

1. Introduction Svabic-Vlahovi 
c, 2004; Stepanovic, Cirkovi 
c, Mijac, & Svabi 
c-
Vlahovic, 2003). The biofilm formation is a stepwise and dynam-
Foodborne diseases cover a wide range of illnesses and are ical process consisting of (i) initial attachment, (ii) irreversible
triggered by agents that were consumed along with food. As stated attachment, (iii) early development of biofilm architecture, (iv)
by the World Health Organization (WHO), the contamination of maturation, and (v) dispersion (Fig. 1).
food may occur at any stage in the process from food production to
consumption (“farm to fork”) and can result from environmental 2.1. Initial attachment
contamination, including the pollution of water, soil or air (WHO,
2007; WHO, n.d). According to the WHO, they are considered an The bacteria’s initial attachment can be active or passive,
emergent public health problem in both developed and developing depending on their motility or the gravitational transportation of
countries (WHO, 2007). The Centers for Disease Control and their planktonic (free floating), diffusion or shear force of the
Prevention (CDC) has stated that foodborne diseases cause surrounding fluid phase (Kumar & Anand, 1998). The cell’s adhesion
approximately 1000 reported disease outbreaks (CDC, 2011) and during this process strongly depends on the physiochemical prop-
a projected 48 million illnesses, 128,000 hospitalizations, and 3000 erties of the bacterial cell surface (Ferreira, Pereira, & Melo, 2010). At
deaths annually in the United States between 1996 and 2010 first, the adherent cells, those that originate biofilm formation on
(Scallan, 2011; Scallan et al., 2011). In addition, many other a surface, possess only a small quantity of extracellular polymeric
outbreaks of pathogens have been found to be associated with substance (EPS) and many are capable of independent movement
biofilms (Aarnisalo, Lundén, Korkeala, & Wirtanen, 2007; Dykes, (O’Toole & Kolter, 1998) by pilus-mediated twitching or gliding
Sampathkumar, & Korber, 2003; Lapidot, Romling, & Yaron, 2006; motility. In this stage, the adhesion is reversible since the attached
Waak, Tham, & Danielsson-Tham, 2002). microorganisms are not yet committed to the differentiation
It is now recognized that biofilms are a frequent source for processda series of morphological changesdwhich leads to biofilm
infections (Costerton, Stewart, & Greenberg, 1999). Around 80% of formation, and many of the cells may detach from the surface and
persistent bacterial infections in the United States were found to be return to the planktonic lifestyle (Stoodley, Sauer, Davies, &
associated with biofilms (Janssens et al., 2008). Since they can Costerton, 2002).
render their inhabitants more resistant to disinfectant (Bower, The surface properties also play an important role in bacterial
McGuire, & Daeschel, 1996; Sidhu, Langsrud, & Holck, 2001), bio- adhesion. Generally, any surface is vulnerable to biofilm develop-
films have become problematic in a wide range of food industries, ment including plastic, glass, metal, wood, and food products. The
including brewing (Flemming & Ridgway, 2009), seafood process- adhesion to the surface is also dependent upon the physicochemical
ing (Shikongo-Nambabi, 2011), dairy processing (Chmielewski & properties of the surface such as texture (rough or smooth) (Donlan,
Frank, 2003), poultry processing (Harvey, Keenan, & Gilmour, 2002), surface charge (Abdallah, Chaieb, Zmantar, Kallel, & Bakhrouf,
2007), and meat processing (Sofos & Geornaras, 2010). 2009), hydrophobicity (Donlan, 2002), pH, temperature (Nilsson,
Much research has been performed to gain deeper under- Ross, & Bowman, 2011), and nutrient composition of the pre-
standing of biofilms and to identify a solution in order to avoid conditioning solution (Donlan, 2002; Gerstel & Römling, 2001). For
contamination of foodstuffs. The objective of this review is to instance, the research of Sinde and Carballo (2000) found that
summarize the problem of biofilms in food industries and the Salmonella and Listeria can attach in a higher numbers to hydro-
current and innovative control strategies that have been used to phobic surfaces than the hydrophilic ones. Furthermore, the surfaces
combat the challenges caused by biofilms. overlaid by a so-called conditioning film comprising macromole-
cules, such as organic substances, will enhance the attachment of
bacterial cells (Tang, Flint, Bennett, Brooks, & Morton, 2009). It has
2. Biofilm and development steps

Biofilms are an aggregation of microorganism attached to and

growing on a surface (Costerton & Stewart, 2001). The formation and
development of biofilms is affected by many factors, including the
specific bacteria strain (Borucki, Peppin, & White, 2003; Chae & Schraft,
2000), material surface properties, and environmental parameters
such as the pH and nutrient levels and temperature (Donlan, 2002).
Biofilm cells are more resistant to antimicrobial agents than planktonic
bacteria, as they have a barrier which prevents or lessens the contact
with antimicrobial agents (O’Toole & Kaplan, 2000).
Any type of microorganisms, including those that cause spoilage Fig. 1. Stages of biofilms development. This diagram is a cartoon of the 5 stages of
and pathogenic microorganisms, could form a biofilm and play biofilm development: 1 reversible attachment, 2 irreversible attachment, 3 early
development of biofilm architecture, 4 maturation and 5 finally dispersion. Under the
a key role in many infections (Parsek & Singh, 2003). Experiencing cartoon are 5 electron micrographs showing what the biofilm actually looks like at
an inhospitable environment, it is important for bacteria to form each stage. Image by D. Davis from Monroe, D “Looking for chinks in the armor of
a biofilm as a survival strategy (Stepanovi c, Cirkovi
c, Ranin, & bacterial biofilms” PLoS Biol, Vol. 5, issue 11.
574 S. Srey et al. / Food Control 31 (2013) 572e585

also been shown that any pre-existing EPS will facilitate the adhe-
sion (Flemming & Schaule, 1988).

2.2. Irreversible attachment

The change from reversible to irreversible attachment is a shift

from a weak interaction of the bacteria with the surface to
a permanent bonding with the presence of EPS (Stoodley, Sauer,
et al., 2002). After irreversible attachment, strong shear force or
chemical breaking of the attachment forces by enzymes, deter-
gents, surfactants, sanitizers (Sinde & Carballo, 2000), and/or heat
is needed for biofilm removal (Augustin, Ali-Vehmas, & Atroshi,
2004; Maukonen et al., 2003; Sinde & Carballo, 2000). Gerke, Kraft,
Süssmuth, Schweitzer, and Götz (1998) worked on Staphylococcus
epidermis and has shown that adherent bacteria produce a poly-
saccharide intercellular adhesion that bonds the cells together and
facilitates microcolony formation and biofilm maturation.

2.3. Early development of biofilm architecture (microcolony

formation)

Microcolony formation results from concurrent accumulation

and growth of microorganisms and is associated with the produc-
tion of EPS (Chmielewski & Frank, 2003), which helps strengthen
the bond between the bacteria and the substratum and stabilizes
the colony from any environmental stress (Donlan, 2002). Studies
of bacterial species in natural systems have shown that accumu-
lation could embrace the recruitment of planktonic cells from the
surrounding medium as a result of cell-to-cell communication
(quorum sensing) (McLean, Whiteley, Stickler, & Fuqua, 1997; Pesci
et al., 1999). O’Toole and Kolter (1998) indicated that in order to
allow microcolony formation of Pseudomonas aeruginosa PA14 to
take place type IV pili are required. Swimming motility was
considered to enable the microorganisms to overcome repulsive
forces at the surfaceewater interface, allowing them to reach the
substratum and form microcolonies by twitching motility powered
by extension and retraction of type IV pili (Skerker & Berg, 2001).
Microcolonies can be very beneficial as they provide interspecies
substrate exchange and/or mutual end-product removal to bacteria
(Costerton, Lewandowski, Debeer, Caldwell, & Korber, 1994).
2.4. Maturation
The biofilm maturation is the step where it develops into an
organized structure which can be flat or mushroom-shaped (Fig. 2),
depending on the nutrient source it is reliant upon (Chmielewski &
Frank, 2003; Klausen et al., 2003). In order to reach structural
maturity, periods of 10 days or more are required (Stoodley, Sauer,
et al., 2002). Bacteria grow under sessile form in heterogeneous
complex-enclosed microcolonies scattered with open water chan-
nels (Davey & O’Toole, 2000). In a study, the mature biofilms was
evaluated and compared with chemostat cultures of P. aeruginosa
by DNA microarray technology (Whiteley et al., 2001). The study
showed that over 70 genes were altered, including genes encoding
proteins involved in translation, metabolism, membrane transport
and/or secretion, and gene regulation.
2.5. Dispersion
Dispersion is the last step in the biofilm formation cycle, and it
allows the cells to revert into their planktonic form (Sauer, Camper,
Ehrlich, Costerton, & Davies, 2002). External perturbation, such as
increased fluid shear (Stoodley, Cargo, Rupp, Wilson, & Klapper,
2002), internal biofilm processes, such as endogenous enzymatic
degradation, or the release of EPS or surface-binding protein, are all
Fig. 2. SEM images of biofilm formation on polystyrene coupons in A. hydrophila.
possible causes of biofilm detachment (Kaplan, Ragunath,
Ramasubbu, & Fine, 2003; Kaplan, Ragunath, Velliyagounder,
Fine, & Ramasubbu, 2004). Detachment seems to be an active
process which allows for the colonization of new niches (Sauer
et al., 2002). In addition, starvation is also considered as a reason
of detachment and allows bacteria to search for a nutrient-rich
environment (O’Toole & Kaplan, 2000).
Other researchers have also proposed the involvement of
enzymatic activity in the degradation of EPS. For instance, the
production of surface protein releasing enzyme dispersin B that
degrades the biofilms of Staphylococcus epidermidis is found in the
Actinobacillus actinomycetemcomitans (Kaplan et al., 2004). Another
study indicates that biofilms can be removed from surfaces by
gentle rinsing if there is an overexpression of alginate lyase which
would cause the degradation of alginate.
3. Biofilm formation
3.1. Biofilm forming strength
The ability to form biofilm varies greatly not just between species.
Even in the same species with different strains and serovars, biofilm
formation strength can vary significantly. For instance, in a study on
the ability of five Salmonella enterica serovars to attach to and colo-
nize intact and cut lettuce and cabbage surfaces (Patel & Sharma,
2010) showed that S. enterica serovars Tennessee and Thompson
can produce considerably more biofilm than serovars Braenderup,
Negev, and Newport; and could be considered as strong biofilm
formers according to the criteria suggested by Stepanovi c et al.
(2004). In another work, Listeria monocytogenes Scott A and 3990
were known to be high biofilm producers, while L. monocytogenes
strains YM96 and 303 were moderate biofilm producers and
L. monocytogenes 17 was the least productive biofilm former
(Chmielewski & Frank, 2006). However, there are also other factors
that influence biofilm forming capacity, specifically surface proper-
ties and nutrient availability. Salmonella spp. and L. monocytogenes
 S. Srey et al. / Food Control 31 (2013) 572e585 575

strains preferably adhere to hydrophobic surface materials (Donlan,
2002). Furthermore, biofilm production of Bacillus cereus on stainless
steel coupons at the aireliquid interface was found to be thicker than
that in submerged systems (Wijman, de Leeuw, Moezelaar,
Zwietering, & Abee, 2007). Similarly, Staphylococcus isolated from
food and food processing environments was studied, and the biofilm
was thicker with the presence of sodium chloride or glucose
(Møretrø et al., 2003). It has also been speculated that carbohydrate
metabolism may have an effect on biofilm production among various
gram-positive bacteria (Pillai et al., 2004). As can be seen, biofilm
formation capacity varies between genera, species, and strains, and it
is influenced by other factors such that one type of bacteria can be
a strong biofilm producer under a certain environment and become
weak in another environment.
3.2. Food contact surface materials for biofilm formation
Besides bacteria genus and species, extrinsic factors also play
a major role influencing the degree of attachment and biofilm
formation. Materials that make up food contact surfaces were
postulated to have a large effect on the level of attachment and bio-
film formation. The materials used for food contact surfaces are
known to be stainless steel, glass, rubber, polyurethane (Chia, Goulter,
McMeekin, Dykes, & Fegan, 2009), Teflon, nitrile butyl rubber (NBR,
Buna-n) (Storgards, Simola, Sjöberg, & Wirtanen, 1999), and
wooddfor developing countries (Mariani et al., 2011). Stepanovic
et al. (2004) reported that Salmonella spp. and L. monocytogenes can
produce high biofilms on plastic surfaces. In another study, the
surface roughness of a polyesterurethane conveyor belt was reported
to have a significant influence on the biofilm forming ability of
L. monocytogenes. Even the weak strains demonstrated the ability to
form biofilm and could not totally be eliminated (Chaturongkasumrit,
Takahashi, Keeratipibul, Kuda, & Kimura, 2011). Adetunji and Isola
(2011) studied the biofilm formation on wood, stainless steel, and
glass surfaces, with the results demonstrating that wood encourages
biofilm formation due to its porosity and absorbency, which can
entrap organic material and bacteria. The authors stated that glass is
the preferred food contact surface owing to its smooth surface and
corrosion resistant properties, while stainless steel can resist the
impact damage better but it is vulnerable to corrosion. Chia et al.
(2009) found that Salmonella attached the best to Teflon, followed
by stainless steel and glass, then Buna-n and polyurethane. It is
suggested that the adhering cells depend on the interfacial free
energies of the surface. Their results suggested that surface rough-
ness has no correlation with adhesion, while many other authors
have stated the opposite (Howell & Behrends, 2006; Scardino,
Harvey, & De Nys, 2006). It has been speculated that the difference
between the degree of surface roughness studied depends on
a subjective assessment (e.g. polished or unpolished), which could be
the reason for the different observations (Chia et al., 2009). Corre-
spondingly, Arnold, Boothe, Suzuki, and Bailey (2004) reported that
there were dramatically less bacterial cells attached to electro-
polished stainless steel compared to an untreated stainless steel
surface. It was also suggested that besides the material itself other
hygienic design such as welding, joints, corners, and equipment
design could also be an important factor affecting biofilm formation
(Guðbjörnsdóttir, Einarsson, & Thorkelsson, 2005).
documented that the safety of prolonging the shelf life of fresh-cut
produce depends on the washing step, while the efficacy of sani-
tizers on produce can only reduce at most 2 log of bacteria; the
microorganisms presumably are protected by their location in the
plant tissue (internalized, in stomata, cracks, crevices, cut surfaces),
and/or by biofilms produced by the microbes themselves (Whipps,
Hand, Pink, & Bending, 2008). Trimming, cutting, washing, rinsing,
dewatering and packaging are all used in produce industry and are
considered to be the primary source of cross-contamination
(Suslow, 2001). López-Gálvez, Gil, Truchado, Selma, and Allende
(2010) investigated the effect of subsequent washing with chlo-
rine or sodium hypochlorite on short-term cross-contaminated
fresh-cut lettuce. The results indicated that subsequent sanitation
steps cannot efficiently inactivate Escherichia coli cells on the
vegetable tissue. Through scanning electron microscopy, bacteria
cells were detected in clusters or tissue stomata (Fig. 3) where they
are considered as protected from disinfectants, which could explain
the inefficiency of disinfecting solutions. However, chlorine dioxide
and sodium hypochlorite solutions were able to inactivate a large
portion of E. coli cells that were transferred from an inoculated
sample to wash water. It has been suggested that the use of sani-
tation solutions may be able to reduce cross-contamination
between clean and contaminated produce during the washing
process. It is recommended that disinfectants should be used in
order to avoid cross-contamination from contaminated produce to
clean produce (Keskinen, Burke, & Annous, 2009; López-Gálvez
et al., 2010).
Produces are generally consumed raw, hence packingdthe last
step of processingdis considered as one of the critical control
points. This last step should be controlled attentively and regularly
to avoid re-contamination. It has been observed that 12.3% of all
foodborne outbreaks from 1990 to 2007 were related to fresh
produce whereas 10% caused by improper handling post harvesting
from farm and the rest were associated with growing, packing,
shipping or processing of fresh produce (Alliance For Food and

4. Biofilm: problems in food industry

4.1. Produce industry

Currently, microbial control strategies are not efficient enough

to provide a complete eradication of hazardous microorganisms
without affecting product qualities (FAO/WHO, 2008, p. 151). It is Fig. 3. Attachment of A. hydrophila on iceberg lettuce stomata grown at 25  C for 24 h.
576 S. Srey et al. / Food Control 31 (2013) 572e585
Farming, 2010). In 2011, there was an outbreak linked to whole
cantaloupe contaminated with L. monocytogenes (Caron 2011). It is
speculated that the root cause of the outbreaks is the unsanitary
condition of the packing shed. Moreover, the microorganisms were
also found other places include the conveyor belt, drying area and
floor drain (Neuman, 2011). It is supposed that pathogens become
firmly attached in inaccessible place (Neuman, 2011) and form
biofilm then internalize within the produce (Sapers, 2001).
Microorganisms are found to preferentially attach to intact
surfaces of produce (Liao & Cooke, 2001). Gandhi, Golding, Yaron,
and Matthews (2001) found evidence of bacteria at a depth of
12 mm within intact alfalfa sprout tissue. In another study by Han,
Sherman, Linton, Nielsen, and Nelson (2000), it was noticed that
there was no remarkable growth of E. coli O157:H7 found on
uninjured green pepper surfaces after inoculation and incubation
for 24 h at 37  C; while significant growth and multiplication was
found on intact surfaces. Sanitizers such as ozone, chlorine, and
organic acid were reported to be ineffective against microbial
biofilm (Ölmez & Temur, 2010). Furthermore, E. coli was reported to
have survived a 20,000 ppm chlorine soak and grew to a level of
6 log CFU/g radish sprouts, which is remarkably higher than the
initial count on the seeds. It was demonstrated that bacteria were
generally detected in the roots of radish sprouts and all across the
surface, and the cells are found to be located in biofilms attached to
radish sprout tissues during sprouting (Fransisca, Zhou, Park, &
Feng, 2011). Hydrogen peroxide is also relatively ineffective in
E. coli 766 elimination on cantaloupes; it was observed to be
reduced by less than 1 log. Furthermore, immersion of cantaloupes
in 1% hydrogen peroxide for 2 h did not show any increase in
efficacy. It is speculated that this inefficacy is a result of the
oxidation of the agents on the melon surface or from access to
endogenous catalase (Sapers & Sites, 2003).
4.2. Dairy industry
Milk is a very perishable product and is truly vulnerable to
contamination by various microorganisms. The major sources of
contaminated milk and milk products are usually considered to
come from improperly cleaned and sanitized equipment (Jessen &
Lammert, 2003; Koutzayiotis, 1992). It is well documented that
the bacteria frequently encountered in the dairy environment
belong to the genus Enterobacter (Salo, Ehavald, Raaska, Vokk, &
Wirtanen, 2006), Listeria (Waak et al., 2002), Lactobacillus, Micro-
coccus, Streptococcus, Bacillus (Sharma & Anand, 2002) and Pseu-
domonas (Wiedmann, Weilmeier, Dineen, Ralyea, & Boor, 2000). In
2011, there was a recall of approximately 20 pounds of raw milk
distributed in Washington state due to L. monocytogenes contami-
nation (Anonymous, 2011). It is speculated that the type of bacteria
in the milk samples may have shown biofilm formation. For
instance, the larger the amount of thermoduric Streptococci and
Bacillus species in pasteurized milk compared to raw milk could be
caused by the contamination of the dispersion of biofilm (Flint,
Bremer, & Brooks, 1997).
To date, there are many studies focused on the various bacteria
in dairy factories. Several studies (Mafu, Roy, Goulet, & Magny,
1990; Vanhaecke et al., 1990) have indicated that there is no
correlation between surface irregularities or roughness and the
bacteria attachment ability. However, Latorre et al. (2010) con-
ducted a study on the presence of L. monocytogenes-containing
biofilm in milking equipment as a potential source of contamina-
tion on a dairy farm. After having obtained positive results, electron
microscopy scanning of the equipment showed the presence of
individual and clusters of bacteria, and it was postulated that it was
mainly associated with scratched surfaces.
In another study, attachment and biofilm formation by
L. monocytogenes on stainless steel (SS, type 304, no. 4 finish) and
Buna-n rubber (BN, acrylonitrile butadiene, 70 durometer), which
are commonly used in food processing equipment, was evaluated
(Helke, Somers, & Wong, 1993). According to the results, lactose had
no effect on the attachment on either surface compared to the
control PBS, while other compounds showed a significance effect
on the attachment. The authors suggested that the repulsion
between negatively charged proteins and bacterial cell surfaces
could to some extent provide an explanation for the decrease in
attachment. A similar study demonstrated that in the presence of
pasteurized whole milk with a relative humidity (RH) of 75%,
L. monocytogenes endured on the BN surface between 25  C and
6  C, and 6  C on the SS surface and growing at 25  C on the SS
surface, both at the same RH. At the same time, the attached cells
decreased over time and the slowest rate of decrease was at 6  C
and 75.5% (Helke & Wong, 1994). It was also documented that the
BN surface had an inhibitory effect on the growth of
L. monocytogenes and several other foodborne pathogens (Helke &
Wong, 1994; Ronner & Wong, 1993). According to Ronner and Wong
(1993), this inhibitory effect varies depending on the nutrient
contents in the environment and at one-fifth the amount of
tryptose-phosphate broth nutrients, there is an increase in lag time.
However, the effect can endure about 20 cycles of Clean-In-Place
(CIP) (Helke & Wong, 1994). Furthermore, it was suggested that
a slow growth rate could improve resistance to antimicrobial
agents (Anwar, Dasgupta, & Costerton, 1990), which can to some
extent explain why biofilms on BN are more resistant to sanitizers
than normally formed biofilms (Wong, 1998).
Biofilm formation depends on many factors in dairy industry.
Dairy products are very susceptible to contamination by biofilms
and it is challenging to eliminate those microorganisms.
4.3. Fish processing industry
In the fish processing industry, both equipment and water
quality are considered to be major concerns. Also, many types of
fish-contaminated-bacteria are found to be biofilm-forming,
including Vibrio cholerae (Faruque et al., 2006), Vibrio para-
haemolyticus (Enos-Berlage, Guvener, Keenan, & McCarter, 2005),
Vibrio vulnificus (Joseph and Wright 2004), and Vibrio alginolyticus
(Kogure, Ikemoto, & Morisaki, 1998). Many genera other than Vibrio,
such as L. monocytogenes, Salmonella spp., Bacillus spp., Aeromonas,
and Pseudomonas spp., are also known to be biofilm forming in fish
and seafood processing (Rajkowski, 2009).
As important as any of the other components used in seafood
industry, seawater is used instead of freshwater for economical
reason. Even though the water is treated in a combined chlorina-
tion and Ultraviolet (UV) radiation system, treated seawater was
found to be contaminated with Vibrio spp., caused by biofilm
formation in the seawater distribution system after the treatment
process (Shikongo-Nambabi, Kachigunda, & Venter, 2010). Various
authors have stated that the failure of chlorine to inhibit biofilm
formation and mature biofilm was not due to the effect of pH
(Momba & Binda, 2002; Shikongo-Nambabi et al., 2010). It has been
established that there is a loss in water quality after undergoing
treatment process, which is usually caused by cells detaching from
the biofilm in the distribution system due to the ineffectiveness of
residual chlorine, and that this is common in water distribution
systems (Momba & Makala, 2004; September, Els, Venter, & Brözel,
2007; Shikongo-Nambabi et al., 2010). Furthermore, biofilm
formation by seafood contaminated Vibrio harveyi on surfacesdbe
it plastic, cement slab or steel coupondwas also evaluated. Results
showed that there was only a slight reduction of biofilm on the
concrete slabs and plastic (chlorine 20 ppm; 10 min), while the cells
 S. Srey et al. / Food Control 31 (2013) 572e585
were completely killed at a higher concentration (chlorine
100 ppm; 10 min) on the steel coupons. This is particularly
important for shrimp hatcheries given that water storage tanks,
polythene water pipes, and surfaces of larval tanks are susceptible
to biofilm formation (Karunasagar, Otta, & Karunasagar, 1996).
In a study by Guðbjörnsdóttir et al. (2005), it was shown that
adhered bacteria are found in many locations on the seafood pro-
cessing lines, despite the fact that thorough cleaning and disin-
fection are carried out regularly. Gram-negative rods, namely
Pseudomonas spp., Aeromonas spp., Enterobacteriaceae, and yeast,
were isolated from a shrimp processing plant, while Pseudomonas
spp. and Enterobacteriaceae were found mainly in the fish pro-
cessing plant. Pseudomonas putida and Pseudomonas fluorescens
were the main species of Pseudomonas spp. isolated from the
shrimp factories. These organisms are considered as the causes of
spoilage in fresh or chilled fish (Gram and Huss 1996). More
importantly, it was noted that the presence of Pseudomonas spp.
would significantly enhance the colonization of L. monocytogenes
on stainless steel (Guðbjörnsdóttir et al., 2005). It was seen that
Vibrio spp. are not the only genus found in seafood processing
plants. Other genera such as Pseudomonas spp., Aeromonas spp., and
so on are also found to be significant biofilm producers and their
presence would enhance the biofilm formation of other genus. In
another study on the correlation of the biofilm forming abilities of
Salmonella and its persistence in fish meal- and feed factories,
Salmonella agona and Salmonella montevideo were found to be good
biofilm producers, while Salmonella typhimurium was found to be
a poor biofilm producer. The results of the study also indicated that
the persistence of Salmonella in the factory environment depends
significantly on the strain’s biofilm forming ability (Vestby,
Møretrø, Langsrud, Heir, & Nesse, 2009). However, it also indi-
cated that the level of biofilm formation can be affected positively
or negatively by environmental factors, such as natural microflora
(Carpentier & Chassaing, 2004; Guðbjörnsdóttir et al., 2005).
4.4. Poultry industry
Many studies have been carried out on the biofilm formation in
the poultry processing industry. Under many investigations, it has
been identified that dust, surfaces, feces, poultry feed, and trans-
portation of live poultry between production and processing units
are known to be the important risk factors in Salmonella contami-
nation (Marin, Hernandiz, & Lainez, 2009; Park, Jarquin, Hanning,
Almeida, & Ricke, 2011; Ramesh, Joseph, Carr, Douglass, &
Wheaton, 2002). Furthermore, approximately 50% of the strains
isolated on poultry farms were able to produce biofilms (Marin
et al., 2009). The attachment of 25 Salmonella strains to four
different materials (polytetrafluoroethylene-also known as Teflon,
stainless steel, rubber, and polyurethane), which are commonly
used in poultry industry, was studied. Among those, Salmonella
sofia isolates (except S1635 and S1636) can generally adhere in
higher numbers to the different surfaces than the other isolates. It
was suggested that S. Sofia serovar may have more pili or fimbriae
than others (Chia et al., 2009). The results of the study also agree
with the data from Helke et al. (1993) that S. typhimurium can
attach more readily to stainless steel than rubber. In a different
study, the characterization of biofilm formation and the attachment
of S. typhimurium DT104 (Kim & Wei, 2009) was evaluated, with the
results indicating that the main contributor to lipopolysaccharide
(LPS) synthesis and biofilm formation of S. typhimurium DT104 is
rfbA gene. According to the study, many factors, such as production
of EPS and their efficient transportation through outer membranes,
expression of flagella and regulation of exoribonucleases and RNA-
binding protein, were suggested to be involved in biofilm formation
and the attachment of S. typhimurium DT104 on contact surfaces.
577
Besides Salmonella spp., Campylobacter spp. are also commonly
found pathogens in poultry and poultry processing (Deming et al.,
1987; Harris, Weiss, & Nolan, 1986; Hopkins & Scott, 1983;
Sanders, Boothe, Frank, & Arnold, 2007). Many researchers have
been trying to understand the behavior of Campylobacter jejuni in
poultry processing. As a result, it has been determined that
temperature is a significant factor influencing their survival (Chan,
Tran, Kanenaka, & Kathariou, 2001; Dykes et al., 2003; Trachoo,
Frank, & Stern, 2002). Other authors have stated that wild-type
cultures of C. jejuni planktonic survive longer at lower tempera-
tures than higher temperatures when faced with multiple stressors
(Chan et al., 2001; Dykes et al., 2003). In contrast, Hanning, Jarquin,
and Slavik (2008) showed that culturable C. jejuni can endure
longer in biofilms at 32  C, compared with survival of culturable
planktonic cells and biofilms at 10  C. The results of the same study
demonstrated that the attachment of C. jejuni to surfaces is facili-
tated by a pre-existing biofilm so it is important to strengthen the
control of biofilms in poultry processing.
4.5. Meat industry
Organic residues in food processing could be a niche for micro-
organism accumulation and biofilm formation as it is a source of
cross-contamination, and has become quite a concern for numerous
researchers (Brooks & Flint, 2008; McLandsborough, Rodriguez,
Pérez-Conesa, & Weiss, 2006; Simões, Simões, & Vieira, 2010).
Many studies have been conducted in order to gain a deeper
understanding of biofilms. For example, E. coli has been studied by
many researchers to investigate the “attachment and biofilm
formation by E. coli O157:H7 at different temperatures, on various
food-contact surfaces encountered in beef processing” (Dourou
et al., 2011). In the results from the study, the cells attached to the
surface increased over time even under low temperatures, which
could result from the migration of cells toward the surface by
flagellar-mediated motility and Brownian motion (Van Houdt &
Michiels, 2005). It has been seen that at low temperatures, micro-
organisms can adhere and survive on food contact surfaces, and
even increase in population as time progressed (Dourou et al., 2011;
Kim, Ryu, & Beuchat, 2006). Another important factor influencing
the attachment of E. coli O157:H7 is the presence of other micro-
organisms on the surfaces (Castonguay et al., 2006; Klayman,
Volden, Stewart, & Camper, 2009; Marouani-Gadri, Augier, &
Carpentier, 2009). As an example, E. coli O157:H7 was found to be
incapable of forming a biofilm under dynamic-flow conditions due
to the shear forces, while Acinetobacter calcoaceticusdisolated from
meat-processing plantsdmonospecies biofilms are heterogeneous,
well organized, and can develop under both static and dynamic
conditions. The research of Habimana, Heir, Langsrud, Asli, and
Møretrø (2010) indicated that E. coli O157:H7 cells were
embedded and cover by a Acinetobacter calcoaceticus biofilm under
both static and dynamic growth conditions. It is now well docu-
mented that multispecies biofilms may increase the opportunities
for pathogens to thrive in the food industry (Burmølle et al., 2006;
Habimana et al., 2010; Stewart & Franklin, 2008).
4.6. Ready-to-eat (RTE) foods industry
Due to lifestyle changes, RTE foods have become very popular.
However, RTE foods can be considered as a relatively high risk food
since the products will be consumed directly without undergoing
any bactericidal process. Even though RTE foods have been well
processed, the chances of being contaminated are relatively high.
Furthermore, the storage times and conditions are known to
be important factors affecting RTE foods quality (Sofos & Geornaras,
2010). RTE foods could potentially be susceptible to cross-
578 S. Srey et al. / Food Control 31 (2013) 572e585
contamination during processing and handling. In particular, after
cooking, the products may be recontaminated during loading,
conveying, weighing, and packaging (Osaili, Alaboudi, & Nesiar,
2011). Garrido et al. (2009) evaluated the occurrence and levels of
L. monocytogenes in refrigerated RTE products for prolonged
consumption in northern Spain. It was found that RTE smoked fish
was the most common pathogen-contaminated food among the
samples analyzed. The slicing materials used in processing were
speculated to be the source of contamination due to biofilm
formation. Comparably, smoked salmon was found to be the most
L. monocytogenes contaminated product in the study by Di Pinto,
Novello, Montemurro, Bonerba, and Tantillo (2010). According to
a survey by Wagner, Auer, Trittremmel, Hein, and Schoder (2007),
fish and seafood were also found to be highly contaminated with
L. monocytogenes among the samples analyzed. The survey also
shows that there is more chance of contamination for unpackaged
or repackaged RTE foods; furthermore, raw meat sausages were
postulated to be potentially contaminated. In addition, the
production of biofilms and quorum sensing by E. coli O157:H7 and
its transfer from contact surfaces to meat, poultry, RTE deli prod-
ucts, and produce products was studied. As a result, the E. coli
O157:H7 strain could be a concern in a wide variety of food prod-
ucts since it can form biofilms on food contact surfaces during food
processing (Silagyi, Kim, Lo, & Wei, 2009).
5. Biofilm control strategies
Since biofilms are a great concern in the food sectors, many
studies have been done in order to gain a better understanding of
their development and spread. Consequently, many studies have
also come up with countermeasures. The first and most important
thing to do is to prevent biofilm formation by regularly cleaning
and disinfecting so as to not allow the cells to firmly attach
(reversible attachment) to contact surfaces (Midelet & Carpentier,
2004; Simões, Simões, Machado, Pereira, & Vieira, 2006). Meyer
(2003) suggested three different strategies: (i) disinfection “in
time”, before biofilm develops, (ii) disinfection of biofilms using
harsh disinfectants, and (iii) inhibition of attachment of microbes
by selecting surface materials that do not promote attachment or
by supplementing with nutrients. Many other researchers have
accounted for the incorporation of antimicrobial products in the
surface materials themselves (Knetsch & Koole, 2011; Park,
Daeschel, & Zhao, 2004) by coating surfaces with antimicrobials
(Knetsch & Koole, 2011; Thouvenin et al., 2003) or by modifying the
surfaces’ physiochemical properties (Chandra et al., 2005;
Rosmaninho et al., 2007). In a study on biofilm control, micropar-
ticles (CaCO3) coated with benzyldimethyldodecylammonium
chloride were found to effectively inactivate biofilm formation
(Ferreira, Pereira, & Pereira, 2011). Many others reported biofilm
formation was inhibited by silver coating surfaces (Hashimoto,
2001; Knetsch & Koole, 2011). Pre-conditioning the surface with
a surfactant has also been reported to prevent bacterial adhesion
(Chen, 2012; Choi, Park, Lee, Park, & Kim, 2011). The research of
Zeraik and Nitschke (2010) demonstrated that after conditioning
with surfactant, the surface became more hydrophilic. The data
illustrated the decrease in hydrophobicity on the treated surfaces
and showed a significant decrease in bacterial attachment.
However, other factors are still considered to contribute to the
reduction of bacterial attachment.
5.1. Cleaning and disinfection
In the food industry, there is debris everywhere which would
promote the accumulation of microorganisms and encourage bio-
film formation. Therefore, regular cleaning is required so as to
prevent the contamination of food products. A good cleaning
process that removes any food residues and other compounds that
may promote bacteria proliferation and biofilm formation is
particularly effective (Simões et al. 2010). Many different chemical
products may be used in cleaning, including surfactants or alkali
products, or used to suspend and dissolve food debris by decreasing
surface tension, emulsifying fats, and denaturing proteins (Forsythe
& Hayes, 1998; Maukonen et al., 2003). Cleaning should be carried
out in a way that can break-up or dissolve the EPS matrix associated
with the biofilms so that disinfectants can gain access to the
bacteria cells (Simões et al. 2006). It is evident that the use of high
temperatures in cleaning can reduce the physical force required,
such as water turbulence and scrubbing (Maukonen et al., 2003); as
well as inactivate biofilm cells at a certain level (Chmielewski &
Frank, 2006). Besides, cleaning only allows the removal of
approximately 90% of the bacteria from the surfaces and does not
kill them. They might later re-attach to other surfaces and form
a biofilm, thus disinfection is indispensible with the intention of
eliminating them (Gram, Bagge-Ravn, Ng, Gymoese, & Vogel, 2007).
Antimicrobial agents are used in the disinfection process so as to
kill microorganisms so that the surface population is reduced along
with microbial growth on the surfaces. However, the effectiveness
of disinfectants is limited by the presence of organic material
including fat, carbohydrates, and protein-based materials. Other
than that, pH, temperature, water hardness, chemical inhibitors,
concentration, and contact time are also important factors influ-
encing disinfectants’ effectiveness (Bremer, Monk, & Butler, 2002;
Cloete, Jacobs, & Brözel, 1998; Kuda, Yano, & Kuda, 2008). There are
many types of disinfectants including chlorine, hydrogen peroxide,
iodine, ozone, peracetic acid (Chmielewski & Frank, 2007).
5.2. Clean-in-Place (CIP)
Clean-in-Place (CIP) is a process allowing a complete system to
be cleaned without dismantling it or the manual involvement of
the operator. It includes jetting and spraying the surfaces or the
circulation of cleaning solutions throughout the plant with an
increased turbulence and flow velocity (Romney, 1990). There are
many factors that can influence CIP efficacy, including the nature
of the biofilm layer, cleaning chemical composition and concen-
tration, time (Boulange-Petermann, Jullien, Dubois, Benezech, &
Faille, 2004; Changani, Belmar-Beiny, & Fryer, 1997), cleaning
temperature (Lelièvre, Faile, & Bénézech, 2001), cleaning flow rate
and hydrodynamic (Lelièvre, Antonini, Faille, & Bénézech, 2002),
and the cleaning surface characteristics (Lelièvre, Legentilhomme,
et al., 2002). Relatively, Walton (2008) also summarized the basic
principles of cleaning to (i) consider the physical nature and
construction of the equipment to be cleaned, (ii) assess the nature
of the soil to be removed; (iii) select a detergent appropriate to
the removal of that soil, (iv) bring the soil and the detergent
together (at the right temperature, under the right conditions of
flow and turbulence, at the right chemical concentration, for the
right period of time), (v) rinse away all traces of detergent and
soil, with the objective of achieving the standard of cleanliness
appropriate to the duty for which the equipment is destined to be
used, (vi) always undertake cleaning as soon as possible after
completion of the production operation, and (vii) when necessary,
undertake a disinfection or sterilization process immediately
before the equipment is returned to processing or production
duties in order to reduce the level of microbiological contamina-
tion to one consistent with the hygienic standard required for that
duty.
Bremer, Fillery, and McQuillan (2006) mimicked the CIP of dairy
biofilms on a laboratory scale to study the effectiveness of the
different caustic and acid wash steps. According to the results, it
 S. Srey et al. / Food Control 31 (2013) 572e585
was postulated that the efficacy of the cleaning conditions con-
ducted may change according to the amount of soiling and cleaning
cycles, as well as the fact that the removal of bacterial biofilms on
surfaces in a dairy manufacturing plant can be improved by using
caustic and nitric additives. In another study, the life cycle of four
CIP methods (conventional alkaline/acid cleaning with hot water
disinfection, one-phase alkaline cleaning with acid chemical
disinfection, enzyme-base cleaning with acid chemical disinfection
and the conventional method with disinfection by cold nitric acid at
pH 2) was evaluated. It was found that the CIP methods with small
volumes and low temperatures, such as enzyme-based cleaning
and one-phase alkaline cleaning, were the most highly recom-
mended alternative methods (Eide, Homleid, & Mattsson, 2003).
The study on biofilm removal of bacterial isolates sampled in the
food industry by enzymes proposed that the implementation of
enzymatic control of bacterial biofilms in the food industry would
present a noteworthy alternative, while the conventional CIP using
chemical agents is not providing satisfactory hygienic results
(Lequette, Boels, Clarisse, & Faille, 2010).
5.3. Chemical-based control
In the study on the effect of mechanical stress on biofilms
challenged by different chemicals, it was stated that chemical
agents would react with the EPS complex which would enhance the
mechanical biofilm removal. The removal rate was significantly
improved after treating the biofilm with chemical agents (Simões,
Pereira, & Vieira, 2005). However, in another study, bacterial cells
were destroyed after being subjected to chemical agents, while the
EPS matrix was left unaffected. The best result was attained by
applying both chemical and mechanical treatment (Exner,
Tuschewitzki, & Scharnagel, 1987). Accordingly, it was suggested
that mechanical treatment cannot remove bacterial cells (Jessen &
Lammert, 2003). As such, it can be postulated that chemical and
mechanical treatment has a synergistic effect and both play
important roles in biofilm and bacterial cell removal.
5.3.1. Sodium hypochlorite
Sodium Hypochlorite (NaClO) is a chemical compound used for
bleaching or disinfection and as such it has been used for dis-
infecting surfaces. It was reported to be an effective disinfectant for
biofilm inactivation (Ozdemir, Buzoglu, Calt, Stabholz, & Steinberg,
2010; da Silva et al., 2011). However, it is known to be more
effective in low pH than alkaline pH environments (Araújo, Lemos,
Mergulhão, Melo, & Simões, 2011). NaClO was reported to be
a potential biofilm antimicrobial agent against Staphylococcus
aureus (Toté, Horemans, Vanden Berghe, Maes, & Cos, 2010),
Prevotella intermedia, Peptostreptococcus miros, Streptococcus inter-
medius, Fusobacterium nucleatum, and Enterococcus faecalis when
compared to other disinfectants used in a study by Spratt, Pratten,
Wilson, and Gulabivala (2001). In a study by Lomander, Schreuders,
Russek-Cohen, and Ali (2004), 50 ppm sodium hypochlorite solu-
tion was able to significantly eliminate biofilm cells compared to
rinsing in water. In the comparison on detaching biofilm removal, it
was not more effective than water in detaching biofilms. Six
percent NaClO was also reported to be able to kill a significant
amount of E. faecalis biofilms, which is statistically better than other
tested agents. The efficiency of NaClO was tested in the elimination
of L. monocytogenes, Pseudomonas fragi and Staphylococcus xylosus
(Norwood & Gilmour, 2000). Consequently, all planktonic cells
were eliminated with an exposure to 10 ppm free chlorine for 30 s,
while it can reduce only 2 log of a L. monocytogenes biofilm with
a concentration of 1000 ppm with an exposure time of 20 min. It
was speculated that the co-cultured biofilm may enhance its
resistance to disinfectants.
579
5.3.2. Hydrogen peroxide (H2O2)
H2O2 is one of the widely used disinfectants due to its highly
oxidizing capacity based on the production of free radicals which
affect the biofilm matrix and has been found to be efficient against
biofilms (de Carvalho, 2007; de Carvalho & da Fonseca, 2007).
Moreover, many studies have shown its efficiency as a disinfectant
against biofilms (Christina, Penna, Mazzola, Maria, & Martins, 2001;
Toté et al., 2010). From the perspective of safety, H2O2 is known to be
a safe solution which does not cause allergic reactions (Rideout,
Teschke, Dimich-Ward, & Kennedy, 2005). Moreover, Kim, Silva,
Chamul, and Chen (2000) showed that H2O2 can be used at a high
concentration without negatively affecting the product quality.
H2O2 was used against four strains of Vibrio spp. in seawater. It
was found to be very effective in inhibiting biofilm formation at
a concentration of 0.05% (500 mg/l). It can also kill mature biofilms
at concentrations between 0.08% and 0.2% (Shikongo-Nambabi
et al., 2010). In this study, the bacteria killing efficiency was
higher than the previous study of Kim et al. (2000). It is postulated
that the different mineral ions present in seawater are essential in
the enhancement of H2O2 reaction against microorganism
(Shikongo-Nambabi et al., 2010). Likewise, it was reported to
completely eradicate biofilms at a concentration of 5% with
a 15 min exposure time (Robbins, Fisher, Moltz, & Martin, 2005).
5.3.3. Ozone
Ozone, a result of oxygen atoms exposed to high-voltage electric
discharge, is a bluish gas with strong odor and potential oxidizing
properties (Horvath, Bilitzky, & Hüttner, 1985, p. 350). It is a potent
antimicrobial agent which can be used against bacteria, fungi,
viruses, protozoa, and bacterial and fungal spores (Khardre, Yousef,
& Kim, 2001). The microorganisms are eradicated by the disruption
or breakdown of the cell envelope, which in turn leads to the
leakage of the cell contents. Cell lysis is a faster inactivation
mechanism than that of other antimicrobial agents where perme-
ation through the cell membrane is indispensable in order to
effectively inactivate the microbe. Due to its mechanism, it is
speculated that it cannot lead to microorganism resistance (Pascual,
Llorca, & Canut, 2007). Many researchers have demonstrated the
efficiency of ozone against bacterial cells and biofilms (Dosti, Guzel-
Seydim, & Greene, 2005; Lagrange, Reiprich, & Hoffmann, 2004). A
study by Tachikawa, Yamanaka, and Nakamuro (2009) on the
disinfection and removal of biofilms by ozone water on P. fluorescens
and P. aeruginosa biofilms showed that by forming biofilms, the
resistibility of the microorganisms against ozone was increased by
3000 and 10 times, respectively. The reason behind the resistance
could be the reaction between ozone and the biofilm matrix
introduced into the environment by the bacteria. However, the
surviving cells were found to be less than 1% after being treated
with ozone.
5.3.4. Peracetic acid
Peracetic acid is a result of the reaction between hydrogen
́
peroxide and acetic acid, or by the oxidation of acethaldehyde. The
mixture has a strong odor and a low pH (2.8) and is usually
produced in concentrations between 5 and 15%. It has been used in
water purification and as a disinfectant (Anonymous, n.d.-a). It is
known as an ideal antimicrobial agent according to its extreme
oxidizing capacity. Furthermore, it cannot be deactivated by cata-
lase and peroxidasedenzymes that degrade H2O2. This agent also
decomposes into safe and environmental friendly residues in food
(acetic acid and hydrogen peroxide), hence it can be applied
without rinsing and its efficacy is not affected by protein residues
(Anonymous, n.d.-b). A study showed that peracetic acid can reduce
L. monocytogenes biofilm adhered for 24 h by 5 log with a concen-
tration of 0.50% w/v (Cabeça, Pizzolitto, & Pizzolitto, 2008).
580 S. Srey et al. / Food Control 31 (2013) 572e585
According to Frank, Ehlers, and Wicker (2003), peracetic acid can
reduce more than 6 log of L. monocytogenes biofilms on stainless
steel in the presence of fat and protein soil with a concentration of
2.0 ml/l and an exposure time of 10 min. Others studies also
showed the effectiveness of peracetic acid against various micro-
organisms (Marques et al., 2007; Salvia, Teodoro, Balducci, Koga-
Ito, & Oliveira, 2011). According to Toté et al. (2010), peracetic
acid eliminated approximately 98% and 99% of viable S. aureus and
P. aeruginosa, respectively, with only 1 min of contact time but not
the biofilm matrix. However, there are also several other studies
which showed that peracetic acid is inefficient or less effective than
other disinfectants against biofilms (Królasik, Zakowska, Krepska, &
Klimek, 2010; Rossoni & Gaylarde, 2000). It is suggested that
aldehydes do not degrade the biofilm matrix, and instead improve
the stability. The biofilm complex needs to be eradicated before
chemical agents can be used effectively (Exner et al., 1987).
5.4. Other approaches of biofilm control
The conventional control strategies are chemical-based,
however, it is possible that microorganisms hold a certain degree
of resistance to such strategies, or may acquire it later through
mutation or genetic exchange. These processes enable the bacteria
to survive and proliferate under higher concentration of disinfec-
tants (Gilbert & Mcbain, 2003; McBain, Rickard, & Gilbert, 2002).
Therefore, new approaches to controlling biofilms in the food
industry have been introduced. Simões et al. (2010) reviewed
potential green strategies, including control using enzyme, phage,
and microbial interactions and metabolite molecules.
5.4.1. Ultrasonication
Ultrasonication is a well known technique used in various food
industry processes namely freezing, cutting, drying, tempering,
bleaching, sterilization, and extraction (Chemat, Zill-e-Huma, &
Khan, 2011). It was reported to also be used as an efficient biofilm
removal method (Oulahal-Lagsir, Martial-Gros, Boistier, Blum, &
Bonneau, 2000a). Oulahal-Lagsir, Martial-Gros, Bonneau, et al.
(2000b) investigated the use of an ultrasonic apparatus on biofilm
removal from stainless steel and polypropylene surfaces. The
apparatus was demonstrated to be able to remove twice as much of
the industrial milk biofilm as the swabbing method on poly-
propylene sheets. However, it has been documented that even
though lower-frequency in sonation is remarkably more efficient
than higher frequency for reducing biofilm cells viability, bacteria
in food industries cannot be solely eliminated using the present
ultrasonic technologies thus combining techniques of ultrasound
with other treatment techniques were recommended (Piyasena,
Mohareb, & McKellar, 2003; Qian, Sagers, & Piti, 1997). Accord-
ingly, the combination of ultrasound and ethylenediaminetetra-
acetic acid (EDTA), and ultrasound and enzymes showed a higher
efficacy in removing biofilms. The results were more promising
against S. aureus than E. coli biofilms in the study. The authors
stated that this method was in agreement with an industrial control
method, i.e. a combined treatment of ultrasound generation in
enzymes preparation restricted to an active chamber area with
a fast and good reproducible recovery compared to other
approaches (Oulahal, Martial-Gros, Bonneau, & Blum, 2007).
Ultrasound was also reported to increase the effectiveness of
antibiotics against biofilm cells (Peterson & Pitt, 2000). Two
processes were observed to be account for the increased efficiency:
i), ultrasound improves the diffusion of oxygen into the biofilm
matrix which allows biofilm cells to become active, and therefore,
affected by antibiotics and ii), the high ratedenhanced by
ultrasounddof antibiotics transported into the complex may
destroy the bacteria before they gain resistance to the agents
(Carmen et al., 2004). Baumann, Martin, and Feng (2009) also
showed a significant effect on biofilm removal on stainless steel
food contact surfaces by combining the use of ozonation and
sonication.
5.4.2. Enzymes
Enzymes are proteins with catalytic activity on a specific
chemical molecule. It can be an important alternative for biofilm
removal in the food industry. However, EPS is a heterogenic matrix
so, in order to degrade the complex, a combination of enzymes is
required (Simões et al. 2010). Lequette et al. (2010) showed that the
enzymes efficiency on biofilm removal may vary according to the
species of bacteria and it can also be enhanced by combining with
surfactants; this suggests that proteins also contribute to the
adhesion of biofilms as proposed by Hinsa and O’Toole (2006). The
research of Molobela, Cloete, and Beukes (2010) indicated that
protease enzymes were effective in the degradation of P. fluorescens
biofilm’s EPS, while amylase enzymes were less effective. It was
suggested that the structural composition of EPS varies even among
bacteria of the same species, with the way they were formulated
and their mode of action, are the reasons for the enzymes ineffi-
ciency. It is well known that a mixture of proteases and amylases is
commonly used to respond to the variety present in a single
biofilm. While proteases hydrolyze the proteins, amylases break the
bond of carbohydrates associated with the complex. Among all
1,4-glycosidic bond cleaving amylases, a-Amylases are frequently
used because of their thermostability; however, they will not
stay active for long as they are calcium metalloenzymes
(Craigen, Dashiff, & Kadouri, 2011). Similarly, a combination of
polysaccharide-hydrolyzing enzymes and oxidoreductases were
recommended for bacteria biofilm removal due to the wide range of
polysaccharide-hydrolyzing enzymes activities which make it
useful for the degradation of biofilms matrix; the bactericidal effect
of oxidoreductases (Johansen, Falholt, & Gram, 1997). In another
study, individual biofilm cleaning efficiencies of pectin esterase,
pectin lyase, and cellulose were shown to be not as effective against
P. fluorescens mature biofilms. However, the efficacy was enhanced
when pronase was used in the treatment process, and after being
treated with the aforementioned enzymes and the detachment-
promoting-agent, pronase, to simultaneously treat with others
enzymes (Orgaz, Neufeld, & Sanjose, 2007). As can be seen, enzy-
matic control against biofilms could be used as a new and improved
environmental friendly alternative strategy according to its
nontoxic characteristics and instability.
5.4.3. Phages
The first application of phages was in the early 20th century for
bacterial infections treatment in Eastern Europe, and have been
shown to decrease biofilm formation (Curtin & Donlan, 2006;
Merril, Scholl, & Adhya, 2003). Unlike chemical-based antimicrobial
agents that can cause corrosion, phages can be a suitable substitute
(Goldman, Starosvetsky, & Armon, 2009), thus it has also been
proposed as a biofilm control method (Curtin & Donlan, 2006).
Bacteriophage spray treatment was suggested to be an alternative
to dipping, brushing, or sponging of the spinach harvester blade on
a chlorine solution according to its compatibility with harvest
sanitation practices (Patel, Sharma, Millner, Calaway, & Singh,
2011). It was documented that phage components and their
assembly synthesis vary according to the host bacterial growth rate
and their amount of protein-synthesizing during the time of
infection; however, it was also reported that even under a glucose-
limited chemostat, T4 phage can effect E. coli biofilms (Corbin,
McLean, & Aron, 2001). In a study on the effect of the phage
philBB-PF7A on P. fluorescens biofilms, the important role of
convection mechanism was discussed. It was said that biofilm cells
 S. Srey et al. / Food Control 31 (2013) 572e585
lysis is more efficient under static conditions than dynamic
conditions. It was also found that fIBB-PF7A could be a remarkable
biological agent according to its biofilm cells lysing capability in
a markedly rapid time (Sillankorva, Neubauer, & Azeredo, 2008).
The same phage was also used to control the dual species biofilm of
P. fluorescens and Staphylococcus lentus, and accounted for the
dramatic decrease in the target bacteria cells (P. fluorescens).
Surprisingly, it was proved that phages can be used to efficiently
reach and lyse their target bacterium in both single and dual
species biofilms notwithstanding the presence of a non-susceptible
host (Sillankorva, Neubauer, & Azeredo, 2010).
Cerca, Oliveira, and Azeredo (2007) studied the susceptibility of
S. epidermidis planktonic cells and biofilms according to the lytic
action of staphylococcus bacteriophage K. The phage K lysis effi-
ciency depends upon the bacteria growth phase. This was also
found to be true in the research of Sillankorva, Oliveira, Vieira,
Sutherland, and Azeredo (2004). Fluorescence Correlation Spec-
troscopy was used to study the diffusion and reaction of bacterio-
phages inside biofilms; the results indicated that bacteriophages
can infiltrate different biofilm complexes, and that generally, they
are immobilized, amplified, and released by a lytic cycle in the
biofilm and interact with their specific binding sites on the hosts
although the lytic activity was not observed (Briandet et al., 2008).
According to Tait, Skillman, and Sutherland (2002), phages and
bacteria can steadily co-exist in biofilms, thus a mixture of phages
and polysaccharide depolymerases and disinfectant was suggested
for a better biofilm control. To address this challenge, bacterio-
phages can be engineered to be able to express a biofilm-degrading
enzyme, which would be a good asset in overcoming the challenges
in controlling biofilms. The engineered phages were reported to be
able to noticeably eradicate bacterial cells in biofilms (approxi-
mately 99.997% removal), as well as the biofilm complex (Lu &
Collins, 2007).
5.5. Hurdle technology
Hurdle technology is a combination of two or more different
control techniques which have been proven to be effective.
However, in order to achieve an effective treatment, the right
combination is needed. It was shown that the combination treat-
ment of NaClO with UV irradiation had a better reduction of food-
borne pathogens in food than single treatment (Ha & Ha, 2011).
DeQueiroz and Day (2007) studied the antimicrobial activity and
effectiveness of a combination of NaClO and hydrogen peroxide
(H2O2) in killing and removing P. aeruginosa biofilms from surfaces.
It showed an increased reduction in the number of cells with only
a short exposure time. The synergistic effect of a combined treat-
ment of biofilms with H2O2 and UV was also shown to be more
effective than the single use of H2O2 by 10 fold, which could
contribute to a more environmental-friendly treatment
(Vankerckhoven et al., 2011). Schurman, Sumner, and Marcy (2001)
tested the effectiveness of H2O2 and demonstrated that its efficacy
was enhanced when there was an increase in temperature. More-
over, an increase in organic acid concentration could also increase
its efficiency. Additionally, the synergistic effect of ozone and
ultrasound was also shown to be efficient for biofilm cell reduction.
However, the amount of cells eradicated was only slightly more
compared to that when ozone was used alone. It was suggested that
the energy efficiency of the combined process shall be studied in
order to have a cost-effective treatment system (Patil, 2010).
Moreover, the efficiency of antibiotics alone and synergizing with
a lytic bacteriophage in the removal of old Klebsiella pneumoniae
biofilms was examined. The findings revealed that phages can be
surprisingly capable in the removal of older biofilms on account of
depolymerase. Then again, the eradication of biofilms was improved
581
when a bacteriophage is used with an antibiotic (Verma, Harjai, &
Chhibber, 2010). Hurdle technology is a very promising new
approach in controlling biofilms in the food industry.
6. Conclusion
Pathogenic microorganisms in biofilms formed in different food
industries settings are a source of food contamination. As the
demand for fresh, RTE and processed foods increases, many studies
are needed to address biofilm removal and disinfectant efficacy in
food industries. Even though up to now the conventional control
strategies are still used and still being developed, a more
economical and environmental friendly control strategy is indis-
pensible to satisfy the need of industrial food safety. It should be
taken into account that disinfection method shall provide a desir-
able cost-effective result and not causing any adverse effect on
human health as well as the environment. Therefore, we highly
recommended hurdle technology since it gives synergistic effect
which reduces the materials and energy consumption. Moreover,
inhibiting biofilms and quorum sensing by natural antimicrobials
would also be an alternative to combat the biofilms problems.
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