**5. Food technologies to control the biofilm formation**

Some food technologies belonging to alternative technologies seem to be successful for preventing the biofilm formation and/or for targeting resistant microorganisms and making them more susceptible to molecular interventions in order to hinder their biofilm formation ability. Among these technologies are included plasma treatments, ultrasound treatments, light-based technologies, pulsed electric fields (PEF), and high hydrostatic pressures. With the exception of ultrasound treatments that can be used to fight against biofilms formed on mechanical parts or pipes, the others are mostly applied for food matrix decontamination.

### **5.1 Plasma treatments**

Plasma is generated when the added energy ionizes a gas, which is composed of ions, neutrals, and electrons. Plasma treatment is a surface treatment that has a low penetration depth and was reported to be effective against biofilms, depending on the type of surface biofilms are formed on, the distance between plasma and surface, and the thickness or the microbial load.

Plasma sources for producing nonthermal plasma at atmospheric pressure are plasma jets, dielectric barrier discharges (DBD), corona discharges, and microwave discharges. Different other characteristics of the plasma have been reported to influence the biofilms' inactivation such as the setup (electrode configuration), the exposure mode, the operating gas, the frequency, the plasma intensity (voltage), and the time of exposure [122].

Researches [123] showed that the efficacy of DBD in-package atmospheric cold plasma (ACP) against *S.* typhimurium, *L. monocytogenes*, and *E. coli* could reach up to 5 log CFU/g after 300 s of treatment at 80 kV. Other researchers [124] studied the

**227**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

effect of ACP on monoculture biofilms (*E. coli*, *S. enterica*, *L. monocytogenes*, and *P. fluorescens*) and mixed culture biofilms (*L. monocytogenes* and *P. fluorescens*) and demonstrated that the latest are more difficult to inactivate than the former ones*. L. monocytogenes* and *P. fluorescens* inoculated as mixed cultures on lettuce were reduced by 2.2 and 4 log CFU/g, respectively, and the biofilms formed at 4°C were

Govaert et al. [122] studied the influence of different plasma characteristics on the inactivation of *L. monocytogenes* and *S.* typhimurium biofilms and showed

highest reduction was obtained for a DBD electrode with He and no O2 in the gas mixture and an input voltage of 21.88 V. A high efficiency of the inactivation of bacterial biofilm was achieved by DBD for low-dose discharges (70 mW/cm2

short treatment times (≤300 s), and the most effective reduction in the number of *S. aureus* cells of 2.77 log was reported after 300 s. *E. coli* biofilm was reduced only

It was shown that ACP is a promising technique but alone cannot achieve complete biofilm inactivation and thus it should be complemented by other surface treatments. Possibility to combine ACP with different biocides such as hydrogen peroxide, sodium hypochlorite, ethylenediaminetetraacetic acid, chlorhexidine, octenidine, and polyhexanide applied before or after the plasma treatment was tested by [126] to reduce biofilms cultivated on titanium discs. Also, Gupta et al. [127] studied the antimicrobial effect of an ACP, plasma jet combined with chlorhexidine, for the sterilization of the biofilms formed by *P. aeruginosa* on

Ultrasound (US) is a form of energy generated by sound waves at frequencies that are too high to be detected by the human hearing (>16 kHz). The US band is also divided into low frequency (16 kHz−1 MHz) and high frequency (>1 MHz)

US was used as biofilm removal method; however, many studies demonstrated

Combination of US with mild heat and slightly acidic electrolyzed water was used to test the inactivation of *B. cereus* biofilms on green leaf surfaces. Slightly acidic electrolyzed water with 80 mg/L treatment for 15 min combined with US of fixed frequency (40 kHz) and acoustic energy density of 400 W/l at 60°C resulted

Synergistic effects were registered also for ultrasound (US; 37 kHz, 380 W for 10–60 min) assisted by peroxyacetic acid (PAA; 50–200 ppm) on reducing

The efficacy of US (37 kHz, 200 W, for 30 min)-assisted chemical cleaning methods (10% alcohols, 2.5% benzalkonium chloride, and 2.5% didecyl dimethyl

of *B. cereus* reference strains ATCC

that it should be complemented by other inactivation methods [129, 130]. For example, [130] demonstrated that US removed a significant amount of *E. coli* and *S. aureus* biofilm, up to 4 times higher compared to the swabbing method. Later on, the same researchers [131] showed that two ultrasonic devices developed failed to completely remove *E. coli* and *S. aureus* biofilms for closed surfaces, but they succeeded in biofilm inactivation on opened surfaces (10 s at 40 kHz). The use of chelating agents such as EDTA completely dislodged *E. coli* biofilm but not significantly improved *S. aureus* biofilm removal. A synergistic effect was achieved when US was combined with enzymes (proteolytic or glycolytic) that demonstrated a 2–3

times higher efficacy in biofilm removal compared to sonication.

in a reduction of ~3.0 and ~3.4 log CFU/cm2

*Cronobacter sakazakii* biofilms on cucumbers [133].

10987 and ATCC 14579 [132].

), but the

) and

that inactivation can vary from 1 log to approximately 3.5 log (CFU/cm2

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

more resistant than the ones formed at 15°C.

by 66.7% [125].

titanium surfaces [128].

bands.

**5.2 Ultrasound-assisted technologies**

*Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

Metal-based NPs (silver, gold, and metal oxides) with antimicrobial activity can be used to create different nanocomposite materials able to prevent bacterial adhesiveness to food-contact surfaces and equipment. Wu and coworkers [114] showed that cysteine dithiothreitol and beta-mercaptoethanol were able to reduce *S. aureus* biofilm formation on polystyrene polymer. Liang and coworkers [115] revealed that silver salt of 12-tungstophosphoric acid NPs (AgWPA-NPs) can be used to develop new materials for preserving foods, since they were able to inhibit *S. aureus* biofilm formation by damaging bacterial cells' membrane. Moreover, genes related to biofilm formation, such as *icaA, sarA*, and *cidA* were shown to be downregulated as a consequence of AgWPA-NPs' application. Naskar and coworkers [116] tested the antibiofilm activity of polyethylene glycol-coupled Ag-ZnO-rGO (AZGP) nanocomposite on both Gram-positive bacteria (*S. aureus* ATCC 25923) and Gram-negative bacteria (*P. aeruginosa* MTCC 2453). These NPs, at a concentration of 31.25 μg/mL, reduced the biofilm formed by *S. aureus* with ~95% and that formed by *P. aeruginosa* with ~93%. Zinc oxide NPs were used for the destruction of the biofilm formed on glass slide by *S. aureus* and *P. aeruginosa* [117]. Titania nanoparticles can be used to prevent the formation of *P. fluorescens* biofilm on the surfaces of TiO2/polystyrene nanocomposite film [118]. It has been shown that nanostructured TiO2 combined with UVA irradiation can be used to destroy *L. monocytogenes* biofilm, while silver NPs at a concentration of 15 μg/mL had the

The ability of two types of superparamagnetic iron oxide (IONs and IONs coated with 3-aminopropyltriethoxysilane) to inhibit biofilm formation by *B. subtilis* was

Some food technologies belonging to alternative technologies seem to be successful for preventing the biofilm formation and/or for targeting resistant microorganisms and making them more susceptible to molecular interventions in order to hinder their biofilm formation ability. Among these technologies are included plasma treatments, ultrasound treatments, light-based technologies, pulsed electric fields (PEF), and high hydrostatic pressures. With the exception of ultrasound treatments that can be used to fight against biofilms formed on mechanical parts or

Plasma is generated when the added energy ionizes a gas, which is composed of ions, neutrals, and electrons. Plasma treatment is a surface treatment that has a low penetration depth and was reported to be effective against biofilms, depending on the type of surface biofilms are formed on, the distance between plasma and

Plasma sources for producing nonthermal plasma at atmospheric pressure are plasma jets, dielectric barrier discharges (DBD), corona discharges, and microwave discharges. Different other characteristics of the plasma have been reported to influence the biofilms' inactivation such as the setup (electrode configuration), the exposure mode, the operating gas, the frequency, the plasma intensity (voltage),

Researches [123] showed that the efficacy of DBD in-package atmospheric cold plasma (ACP) against *S.* typhimurium, *L. monocytogenes*, and *E. coli* could reach up to 5 log CFU/g after 300 s of treatment at 80 kV. Other researchers [124] studied the

capacity to inhibit *S. aureus* and *E. coli* biofilms [119, 120].

**5. Food technologies to control the biofilm formation**

pipes, the others are mostly applied for food matrix decontamination.

surface, and the thickness or the microbial load.

successfully tested by [121].

**5.1 Plasma treatments**

and the time of exposure [122].

**226**

effect of ACP on monoculture biofilms (*E. coli*, *S. enterica*, *L. monocytogenes*, and *P. fluorescens*) and mixed culture biofilms (*L. monocytogenes* and *P. fluorescens*) and demonstrated that the latest are more difficult to inactivate than the former ones*. L. monocytogenes* and *P. fluorescens* inoculated as mixed cultures on lettuce were reduced by 2.2 and 4 log CFU/g, respectively, and the biofilms formed at 4°C were more resistant than the ones formed at 15°C.

Govaert et al. [122] studied the influence of different plasma characteristics on the inactivation of *L. monocytogenes* and *S.* typhimurium biofilms and showed that inactivation can vary from 1 log to approximately 3.5 log (CFU/cm2 ), but the highest reduction was obtained for a DBD electrode with He and no O2 in the gas mixture and an input voltage of 21.88 V. A high efficiency of the inactivation of bacterial biofilm was achieved by DBD for low-dose discharges (70 mW/cm2 ) and short treatment times (≤300 s), and the most effective reduction in the number of *S. aureus* cells of 2.77 log was reported after 300 s. *E. coli* biofilm was reduced only by 66.7% [125].

It was shown that ACP is a promising technique but alone cannot achieve complete biofilm inactivation and thus it should be complemented by other surface treatments. Possibility to combine ACP with different biocides such as hydrogen peroxide, sodium hypochlorite, ethylenediaminetetraacetic acid, chlorhexidine, octenidine, and polyhexanide applied before or after the plasma treatment was tested by [126] to reduce biofilms cultivated on titanium discs. Also, Gupta et al. [127] studied the antimicrobial effect of an ACP, plasma jet combined with chlorhexidine, for the sterilization of the biofilms formed by *P. aeruginosa* on titanium surfaces [128].

### **5.2 Ultrasound-assisted technologies**

Ultrasound (US) is a form of energy generated by sound waves at frequencies that are too high to be detected by the human hearing (>16 kHz). The US band is also divided into low frequency (16 kHz−1 MHz) and high frequency (>1 MHz) bands.

US was used as biofilm removal method; however, many studies demonstrated that it should be complemented by other inactivation methods [129, 130]. For example, [130] demonstrated that US removed a significant amount of *E. coli* and *S. aureus* biofilm, up to 4 times higher compared to the swabbing method. Later on, the same researchers [131] showed that two ultrasonic devices developed failed to completely remove *E. coli* and *S. aureus* biofilms for closed surfaces, but they succeeded in biofilm inactivation on opened surfaces (10 s at 40 kHz). The use of chelating agents such as EDTA completely dislodged *E. coli* biofilm but not significantly improved *S. aureus* biofilm removal. A synergistic effect was achieved when US was combined with enzymes (proteolytic or glycolytic) that demonstrated a 2–3 times higher efficacy in biofilm removal compared to sonication.

Combination of US with mild heat and slightly acidic electrolyzed water was used to test the inactivation of *B. cereus* biofilms on green leaf surfaces. Slightly acidic electrolyzed water with 80 mg/L treatment for 15 min combined with US of fixed frequency (40 kHz) and acoustic energy density of 400 W/l at 60°C resulted in a reduction of ~3.0 and ~3.4 log CFU/cm2 of *B. cereus* reference strains ATCC 10987 and ATCC 14579 [132].

Synergistic effects were registered also for ultrasound (US; 37 kHz, 380 W for 10–60 min) assisted by peroxyacetic acid (PAA; 50–200 ppm) on reducing *Cronobacter sakazakii* biofilms on cucumbers [133].

The efficacy of US (37 kHz, 200 W, for 30 min)-assisted chemical cleaning methods (10% alcohols, 2.5% benzalkonium chloride, and 2.5% didecyl dimethyl ammonium chloride) for the removal of *B. cereus* biofilm from polyurethane conveyor belts in bakeries using US was better compared to each individual method as demonstrated by [134].

### **5.3 Combined light-based technologies**

Ultraviolet (UV) light technology is based on the emission of radiation within the ultraviolet region (100–400 nm). The antimicrobial behavior of UV light is based on the formation of DNA photoproducts that inhibit transcription and replication and can lead to cell death [135]. Since the absorption of the DNA is in the 200–280 nm range with the maximum at 254 nm, this wavelength of the UV-C range is called germicidal UV light [136].

Pulsed light (PL) is the next-generation approach to UV delivery. PL is a technology that can be used to decontaminate surfaces by generating short-time high-energy light pulses (millions or thousands of a second) of an intense broad spectrum (200–1100 nm). PL can be used to decontaminate a great variety of foods as well as to decontaminate contact surfaces, thus improving safety in foods and extending their shelf life [137]. The antimicrobial effect is based on strand breaks that lead to the destruction/chemical modification of the DNA and thus prevent the replication of the bacterial cell [138].

Recently, Rajkovic and coworkers [139] evaluated the efficacy of pulsed UV light treatments to reduce *S.* typhimurium, *E. coli* 0157:H7, *L. monocytogenes*, and *S. aureus* on the surface of dry fermented salami inoculated with 6.3 log CFU/g at 3 J/ cm2 (1 pulse) or 15 J/cm2 (5 pulses) for 1 or 30 min. The authors found a significant effect of PL treatment time, with the best results after 1 min of applying PL (2.18–2.42 log CFU/g reduction), while after 30 min, the reduction varied from 1.14 to 1.46 log CFU/g.

A comprehensive review in the literature underlined the various researches directed mainly at inactivation of pathogens in food or on surfaces and for preventing biofilm formation [137]. While there are often considerable differences in the rate of microbial inactivation by PL, a maximum reduction of 3-log was typically achieved, which is below the reduction performance standard of 5-log required by HACCP regulation [138].

Regarding the combined methods, synergistic interaction between gallic acid and UV-A light was able to inactivate *E. coli* O157:H7 in spinach biofilm [140]. The UV-A treatment complemented by the gallic acid presence was found to be effective producing a 3-log (CFU/mL) reduction in *E. coli* O157:H7 on the surface of spinach leaves.

However, PL technology limitation related to the inability to effectively treat uneven food surfaces with crevices, the presence of organic material, and large microbial populations generating shading effects should also be taken into account. Future innovation in PL technology will seek to improve fluence efficiency, for example by considering alternative light sources such as LEDs [141], reflective surfaces included in the treatment chamber, using materials such as titanium dioxide to augment irradiation efficacy [138], and other combination of treatments assisted by PL, based on hurdle approach.

### **5.4 Pulsed electric field**

Pulsed electric field (PEF) is a food processing technology that applies short, high-voltage pulses, across a food material placed between two or more electrodes. The pulses enhance cell permeability by damaging the cell membrane, and if the transmembrane potential is sufficiently high, it produces electroporation. Further, if pores are not resealed, it results in cell death. Most of the food applications are

**229**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

in-between the electrodes area that applies the PEFs [142].

designed for liquid flow through pipes where in a certain region the liquid passes

PEF caused a maximum of 66% inactivation, while sublethally injuring approxi-

triethyl citrate) to inactivate *Salmonella* serovars in whole liquid eggs [144].

Thermosonication (TS) was investigated in combination with PEF to determine its effects on inactivation and sublethal injury of *P. fluorescens* and *E. coli*. While TS was applied at a low (18.6 mm) and high (27.9 mm) wave amplitude, PEF was

PEF demonstrated synergistic potential in combination with additives (EDTA or

There is a lot of potential demonstrated by PEF and the combination with different other hurdles could contribute to the elimination of persistent clones able to

High pressure processing (HPP) is a cutting-edge technology that represents an alternative to conventional processing. HPP has the ability to inactivate microorganisms and enzymes and has a minimal impact on sensorial and nutritional

Combined with other different hurdles, the pressure-assisted processing could be oriented toward a more targeted inactivation of pathogens and prevention of

Recent studies were focused on *L. monocytogenes*, a pathogen able to form surface-attached communities that have high tolerance to stress. In order to understand how *agr* gene regulates virulence and biofilm formation, a recent molecular study [147] was conducted. *L. monocytogenes* EGD-e Δ*agrD* showed reduced levels

However, *L. monocytogenes* mutant deficient in *agr* peptide sensing showed no impaired resistance to HPP treatment at 200, 300, and 400 MPa for 1 min compared to wild-type and *L. monocytogenes* EGD-e and thus demonstrating that weakened resistance to cell wall stress is not responsible for the reduced biofilm-

Understanding better the molecular mechanisms of stress-related genes will allow to better target pathogen inactivation and to select the right hurdle combination and parameters of unconventional technologies to able to reduce the susceptibility of certain pathogens to form biofilms. These types of studies are just at the beginning and many more researches are expected to focus on these topics in the

Pathogenic and toxigenic bacteria are able to form biofilms, structures that protect the cells and allow them to remain postsanitation in the food processing

and protect the cells and for quorum sensing communication.

Specific genes are expressed in all the steps of biofilm formation or are upregulated under influence of different biotic or abiotic factors. Genes codify for cell surface structures and appendages (flagella, curli, fimbriae, and pili) that are facilitating biofilm formation by helping bacteria to move toward surfaces and to adhere to them, for extracellular polymeric substances that stabilize the biofilms

of surface-attached biomass in 0.1 BHI (brain heart infusion) broth.

) and high electrical field strength (32 kV cm<sup>−</sup><sup>1</sup>

). TS/

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

mately 26% of the *E. coli* population [143].

applied at a low (29 kV cm<sup>−</sup><sup>1</sup>

**5.5 High pressure processing**

properties of food [145, 146].

biofilm formation.

forming ability.

near future.

**6. Conclusions**

environment.

form biofilms.

### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

designed for liquid flow through pipes where in a certain region the liquid passes in-between the electrodes area that applies the PEFs [142].

Thermosonication (TS) was investigated in combination with PEF to determine its effects on inactivation and sublethal injury of *P. fluorescens* and *E. coli*. While TS was applied at a low (18.6 mm) and high (27.9 mm) wave amplitude, PEF was applied at a low (29 kV cm<sup>−</sup><sup>1</sup> ) and high electrical field strength (32 kV cm<sup>−</sup><sup>1</sup> ). TS/ PEF caused a maximum of 66% inactivation, while sublethally injuring approximately 26% of the *E. coli* population [143].

PEF demonstrated synergistic potential in combination with additives (EDTA or triethyl citrate) to inactivate *Salmonella* serovars in whole liquid eggs [144].

There is a lot of potential demonstrated by PEF and the combination with different other hurdles could contribute to the elimination of persistent clones able to form biofilms.

### **5.5 High pressure processing**

*Bacterial Biofilms*

as demonstrated by [134].

**5.3 Combined light-based technologies**

range is called germicidal UV light [136].

replication of the bacterial cell [138].

(1 pulse) or 15 J/cm2

HACCP regulation [138].

**5.4 Pulsed electric field**

to 1.46 log CFU/g.

ammonium chloride) for the removal of *B. cereus* biofilm from polyurethane conveyor belts in bakeries using US was better compared to each individual method

the ultraviolet region (100–400 nm). The antimicrobial behavior of UV light is based on the formation of DNA photoproducts that inhibit transcription and replication and can lead to cell death [135]. Since the absorption of the DNA is in the 200–280 nm range with the maximum at 254 nm, this wavelength of the UV-C

Pulsed light (PL) is the next-generation approach to UV delivery. PL is a technology that can be used to decontaminate surfaces by generating short-time high-energy light pulses (millions or thousands of a second) of an intense broad spectrum (200–1100 nm). PL can be used to decontaminate a great variety of foods as well as to decontaminate contact surfaces, thus improving safety in foods and extending their shelf life [137]. The antimicrobial effect is based on strand breaks that lead to the destruction/chemical modification of the DNA and thus prevent the

Recently, Rajkovic and coworkers [139] evaluated the efficacy of pulsed UV light treatments to reduce *S.* typhimurium, *E. coli* 0157:H7, *L. monocytogenes*, and *S. aureus* on the surface of dry fermented salami inoculated with 6.3 log CFU/g at 3 J/

cant effect of PL treatment time, with the best results after 1 min of applying PL (2.18–2.42 log CFU/g reduction), while after 30 min, the reduction varied from 1.14

A comprehensive review in the literature underlined the various researches directed mainly at inactivation of pathogens in food or on surfaces and for preventing biofilm formation [137]. While there are often considerable differences in the rate of microbial inactivation by PL, a maximum reduction of 3-log was typically achieved, which is below the reduction performance standard of 5-log required by

Regarding the combined methods, synergistic interaction between gallic acid and UV-A light was able to inactivate *E. coli* O157:H7 in spinach biofilm [140]. The UV-A treatment complemented by the gallic acid presence was found to be effective producing a 3-log (CFU/mL) reduction in *E. coli* O157:H7 on the surface of spinach leaves. However, PL technology limitation related to the inability to effectively treat uneven food surfaces with crevices, the presence of organic material, and large microbial populations generating shading effects should also be taken into account. Future innovation in PL technology will seek to improve fluence efficiency, for example by considering alternative light sources such as LEDs [141], reflective surfaces included in the treatment chamber, using materials such as titanium dioxide to augment irradiation efficacy [138], and other combination of treatments assisted by PL, based on hurdle approach.

Pulsed electric field (PEF) is a food processing technology that applies short, high-voltage pulses, across a food material placed between two or more electrodes. The pulses enhance cell permeability by damaging the cell membrane, and if the transmembrane potential is sufficiently high, it produces electroporation. Further, if pores are not resealed, it results in cell death. Most of the food applications are

(5 pulses) for 1 or 30 min. The authors found a signifi-

Ultraviolet (UV) light technology is based on the emission of radiation within

**228**

cm2

High pressure processing (HPP) is a cutting-edge technology that represents an alternative to conventional processing. HPP has the ability to inactivate microorganisms and enzymes and has a minimal impact on sensorial and nutritional properties of food [145, 146].

Combined with other different hurdles, the pressure-assisted processing could be oriented toward a more targeted inactivation of pathogens and prevention of biofilm formation.

Recent studies were focused on *L. monocytogenes*, a pathogen able to form surface-attached communities that have high tolerance to stress. In order to understand how *agr* gene regulates virulence and biofilm formation, a recent molecular study [147] was conducted. *L. monocytogenes* EGD-e Δ*agrD* showed reduced levels of surface-attached biomass in 0.1 BHI (brain heart infusion) broth.

However, *L. monocytogenes* mutant deficient in *agr* peptide sensing showed no impaired resistance to HPP treatment at 200, 300, and 400 MPa for 1 min compared to wild-type and *L. monocytogenes* EGD-e and thus demonstrating that weakened resistance to cell wall stress is not responsible for the reduced biofilmforming ability.

Understanding better the molecular mechanisms of stress-related genes will allow to better target pathogen inactivation and to select the right hurdle combination and parameters of unconventional technologies to able to reduce the susceptibility of certain pathogens to form biofilms. These types of studies are just at the beginning and many more researches are expected to focus on these topics in the near future.
