Ultrasound-involved emerging strategies for controlling foodborne microbial biofilms

Trends in Food Science & Technology 96 (2020) 91–101

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Trends in Food Science & Technology journal homepage: www.elsevier.com/locate/tifs

Ultrasound-involved emerging strategies for controlling foodborne microbial biofilms

T

Hang Yua,b,c,∗, Yang Liua,b,c, Lu Lia,b,c, Yahui Guoa,b,c, Yunfei Xiea,b,c, Yuliang Chenga,b,c, Weirong Yaoa,b,c,∗∗ a

State Key Laboratory of Food Science and Technology, Jiangnan University, No.1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, China School of Food Science and Technology, Jiangnan University, No.1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, China c Joint International Research Laboratory of Food Safety, Jiangnan University, No.1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, China b

A R T I C LE I N FO

S T R UC T UR E D A B S T R A C T S

Keywords: Foodborne microbial biofilm Emerging strategies Ultrasound Chemical disinfectants Synergistic effect

Background: Pollution of foodborne microbial biofilms is a serious problem in the food industry. Microorganisms in the biofilms become insensitive to environmental stresses and increase tolerance to antimicrobial agents, therefore, making them extremely hard to be inactivated by conventional methods. Ultrasound-involved emerging strategies offer options for effectively controlling the biofilms formed on either food contact surfaces or real foods. Scope and approach: This review emphasizes the significances of either ultrasonication alone or combined with other strategies for controlling foodborne microbial biofilms. Key findings and conclusions: Ultrasound as an emerging technology would effectively destroy biofilm structure and partially inactivate microorganisms in the biofilms; however, stimulated the growth of microbes may happen after treatment of low-frequency and low-intensity ultrasound. Combined ultrasound (especially lowfrequency and high-intensity ultrasound) and chemical disinfectants shows a synergistic effect with a relatively high proportion of inactivated microbes in the biofilms compared with that adopted one strategy alone. Ozone and electrolyzed water are also developed for inactivating microbes and removing the biofilms after combining with the ultrasound. Combined treatment of ultrasonication and chelating agents or enzymes is proved to effectively remove the biofilms instead of achieving a strong bactericidal effect. Mechanical oscillation, localized high temperature and pressure, as well as free radicals generated by cavitation during the ultrasonication can partially destroy the basic structure of biofilms, and furthermore, increase the penetration and diffusion of chemicals into the deeper layer of biofilms for achieving a synergistic effect on the biofilm control.

1. Introduction Biofilms are defined as a structured community of microbial cells enclosed in a self-produced matrix composed of extracellular polymeric substances (EPS), and then adherent to each other and/or to interfaces or surfaces (Costerton, Lewandowski, Caldwell, Korber, & Lappin-Scott, 1995). Such a structure provides a shelter for microorganisms inside the biofilm that increases the protection from antibiotics and mechanical forces. Virulence of microorganisms may be also modulated after forming the biofilm (Savage, Chopra, & Neill, 2013). Since the concept of biofilm was first raised more than 70 years ago, countless problems brought by foodborne microbial biofilms have been identified in the

food industry, e.g. dairy processing, poultry processing, brewing, etc. (Fysun et al., 2019; García-Sánchez et al., 2019; Maifreni et al., 2015). Formation of microbial biofilms is detrimental in many food industrial processes where easily cause contamination of food products and damage for reactors, pipelines, storage tanks, etc. (Murphy, Edwards, Hobbs, Shepherd, & Bezombes, 2016). Chemical disinfectant treatment as one of the conventional strategies has been widely adopted in the food industry for overcoming microbial pollution. However, it should be noted that cells in the biofilms are much more resistant to the chemical disinfectants compared with their planktonic counterparts. The chemical disinfectants are harder to penetrate and diffuse into the biofilms and further inactivate inner cells.

∗

Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, No.1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, China. ∗∗ Corresponding author. School of Food Science and Technology, Jiangnan University, No.1800 Lihu Avenue, Wuxi, Jiangsu Province, 214122, China. E-mail addresses: [email protected] (H. Yu), [email protected] (W. Yao). https://doi.org/10.1016/j.tifs.2019.12.010 Received 15 July 2019; Received in revised form 21 October 2019; Accepted 14 December 2019 Available online 19 December 2019 0924-2244/ © 2019 Elsevier Ltd. All rights reserved.

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pullulans, etc. Food factories, in particular, are very common to form foodborne microbial biofilms because of high nutrients, high moisture, suitable pH, and temperature conditions. Most of the contamination in food products would always be related to pollutions of biofilm as universe phenomena (Cappitelli, Polo, & Villa, 2014). There are increasing biofilm-related pollutions in the food factories, e.g. accumulation of biofilms on surfaces of foods and packaging materials, colonization in equipment and convey belts, and fouling of pipes, heat exchangers, and many harborage sites including crevices, cracks, corners, dead ends, gaskets, joints, and valves (Brooks & Flint, 2008). Therefore, hygiene design as a pre-requirement should be properly done first in the food factories, followed by scheduled cleaning, disinfection, and proper maintenance of equipment (Møretrø, Heir, Nesse, Vestby, & Langsrud, 2012). One of the remarkable features of foodborne microorganisms in biofilms is insensitivity to environmental stresses. The resistance of biofilm cells would become 10 to 10,000 times higher compared with their planktonic counterparts depending on the growing stages of biofilm cells, and types of stress. An example of Salmonella enteritidis biofilm formed in wheat flour as a model for low-moisture food was reported to be thermal resistance (D80 °C, 0.45 aw = 14.1 ± 0.6 min), which was significantly higher than that of the planktonic cell (D80 °C, 0.45 aw = 6.0 ± 0.2 min). The authors further concluded that the growth condition of S. enteritidis biofilm conferred more thermal resistance at a low-moisture environment (Villa-Rojas et al., 2017). Increased tolerance to antimicrobial agents is another character of biofilm. Due to the presence of EPS as a diffusion barrier, it is hard for antibiotics or disinfectants to penetrate through the EPS, and these chemicals can also interact with the exopolymers directly. Therefore, only a limited content of antibiotics or disinfectants would affect the inner cells (Davies, 2003). Moreover, the possible exchange of certain resistance genes among the inner cells would also reinforce their resistances (Ma & Bryers, 2013). Biofilms continuously pose a big concern to the food manufactures due to the persistence of microbial contamination where cleaning of the manufacturing plant becomes ineffective (Sharma & Anand, 2002). Commercial sanitizers or detergents coupled with recommended operating procedures may not effectively resolve the pollution of foodborne microbial biofilms. A previous assessment of 21 commercial sanitizers has shown that all the sanitizers achieved 5 log10 CFU/mL-reduction viable cell of planktonic L. monocytogenes at the manufacturers’ recommended concentration; however, only 3 of the 21 sanitizers achieved a 5 log10 CFU/mL-reduction when treated its biofilm counterpart (Cruz & Fletcher, 2012). Another research on studying C. jejuni in a mixed-culture biofilm isolated from the meat plants indicated that the presence of biofilm significantly enhanced the attachment and survival of inner cells. Although the C. jejuni biofilm was susceptible to 4 sanitizers, C. jejuni was unable to be completely inactivated after the treatment of 3 sanitizers at the highest concentration (200 ppm) and the longest duration (45 s) adopted in this study (Trachoo & Frank, 2002). The results of both studies emphasized that the selection of suitable sanitizers should be very cautious for treating different types of biofilms. Some other strategies, such as adopting an effective cleansing program and combining chemicals with emerging technologies are necessary to ensure the inactivation and removal of foodborne microbial biofilms.

Such a biofilm insusceptibility is considered as a tolerance that is induced by a physiological adaption to the biofilm mode of life (Bridier, Briandet, Thomas, & Dubois-Brissonnet, 2011). Mechanical treatments, such as brushing, scrubbing, flushing, etc. are also commonly adopted in the food industry, some of which may not successfully remove the biofilms or even lead to more hygiene problems. For example, highpressure liquids are normally applied to flush surfaces of equipment and floors but should be concerned about spreading survived microorganisms via aerosols (Branck, Hurley, Prata, Crivello, & Marek, 2017; Meyer, 2003). Mechanical brushing and scrubbing may damage surfaces of equipment and increase their roughness, which make microorganisms easily hidden in cracks and crevices or embedded in the biofilms; therefore, they become sources of recontamination of products during food processing (Hilbert, Bagge-Ravn, Kold, & Gram, 2003). For overcoming the drawbacks of conventional methods as mentioned above, emerging strategies are developing gradually for controlling foodborne microbial biofilms. Among these, ultrasound is believed to be a promising technology for effectively detaching the biofilms from contact surfaces, and leading to a bactericidal effect simultaneously (Bigelow, Northagen, Hill, & Sailer, 2009; Zips, Schaule, & Flemming, 1990). Furthermore, the ultrasonic treatment enhances penetration and diffusion of chemical disinfectants through the microbial biofilms making it suitable to become an auxiliary technology combined with the chemicals treatment or other technologies (Torlak & Sert, 2013). However, a negative aspect of sonic or ultrasonic treatments has been described as a formidable enhancer of bacterial viability after treating the microbial biofilms (Erriu et al., 2014; Murphy et al., 2016). Therefore, aims of present work are to analyze current studies regarding sonic or ultrasonic treatments on the foodborne microbial biofilms, and further to conclude findings on biofilm control by adopting sonication/ultrasonication alone or other ultrasound-involved emerging strategies. Corresponding details of the treatments and mechanisms are to be summarized. Future aspects of ultrasound-involved emerging strategies are to be proposed accordingly. 2. Foodborne microbial biofilms in the food industry Biofilm is a ubiquitous life mode for many microbes that easily form in any place with suitable conditions. It is a dynamic procedure with sequential manners for the formation of foodborne microbial biofilm on contacting foods, equipment, and processing surfaces that involves three main steps, i.e. attachment, maturation, and dispersal (Simões, Simões, & Vieira, 2010). Planktonic foodborne microbes first move near the contact surfaces and start a reversible adhesion by macromolecules in bulk liquid or those coated on the contact surfaces. The planktonic foodborne microbes are continuously transported and absorbed from the bulk liquid to the surfaces. Such an adhesion process gradually becomes irreversible when consolidation of loosely bound microbes is finished by secreting and producing sufficient exopolymers and adhesins. Since the attachment of microbes to a substratum has been done, the following steps start subsequently, including cell growth, replication, microcolonies formation, and EPS accumulation, etc. Additional microbes from the local environment are also possibly recruited simultaneously, and eventually form multispecies biofilms. It is an optimal environment provided by the biofilm which protects inner cells, exchanges genetic materials, and achieves a cell-to-cell communication defined as quorum sensing (QS). The QS systems are capable of regulating many physiological activities, such as biofilm formation, virulence expression and dispersal (Zhang et al., 2018). The dispersal refers to cells which detach from matured biofilms, translocate to new places, and repeat the previous procedures to form new biofilms. There are many foodborne microbes which have relatively strong biofilm-forming ability, e.g. Pseudomonas fluorescence, Staphylococcus aureus, Salmonella typhimurium, Listeria monocytogenes, Escherichia coli, Bacillus cereus, Campylobacter jejuni, Pichia pastoris, Aureobasidium

3. Ultrasound Ultrasound refers to any sound at a frequency level above 20 kHz beyond the threshold of human hearing. Ultrasound waves typically contain compression and expansion cycles. Positive pressure can push molecules together in the compression cycle; and the expansion cycle generates cavities because of a large negative pressure which overcomes liquid's tensile strength (Yu, Seow, Ong, & Zhou, 2018). With the progress of ultrasonic processing, the cavities keep absorbing acoustic 92

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from 6.9 to 5.5 Log10 CFU/mL (Na-Young, Seok-Won, & Sang-Do, 2014). Another study on L. monocytogenes biofilm formed on a polyvinyl chloride drain pipe was detected a similar number of viable cells in the biofilm after a 30 s-ultrasonic treatment (20 kHz and 750 W) compared with the control (Berrang, Frank, & Meinersmann, 2008). Inconsistent results of bactericidal effects in L. monocytogenes biofilm treated by ultrasound may be mainly attributed to a different frequency and intensity/power of ultrasound, and different resistances among microbial strains chosen in the four studies. Mechanisms of bactericidal effects brought by the low-frequency and high-intensity ultrasound are concluded as shown in Fig. 1C. Mechanical oscillation generated by the ultrasound shows no significant difference in the bactericidal effect between Gram-positive or Gramnegative bacteria at the same level. The killing rate of four bacteria and a fungus was proportional to the duration and intensity level of ultrasonic treatment at 26 kHz (Scherba, Weigel, & Brien, 1991). Besides the mechanical oscillation, an extremely high, albeit momentary temperature and pressure environment generated by ultrasound would trigger a thermal effect for further inactivation of microbes in the biofilms (Yu, Keh, Seow, Ong, & Zhou, 2017). As another result of cavities’ implosion after absorbing sufficient energy, the sudden reversal in the motion of bubble wall produces a shock wave and high temperature, which can fragment water and other molecules into free radicals. For the fragment of water, free radicals with high reactivity are first generated, e.g. H· and ·OH, and further generate hydrogen peroxide (H2O2) as a strong bactericide (Joyce, Phull, Lorimer, & Mason, 2003). Moreover, the localized heating and free radicals are capable of attacking EPS and cell membranes (Piyasena, Mohareb, & McKellar, 2003). Therefore, the destruction of EPS in the biofilms, and the change of permeability and integrity of cell membranes are consequences after the ultrasonic treatment. A study on evaluating Pseudomonas aeruginosa biofilm after an ultrasonic treatment (70 kHz, 4.6 W/cm2) indicated that some perturbations and holes were found in the outer membrane lipid bilayer, which would transport relatively large hydrophilic compounds and leak out of inner metabolites and ions (Runyan et al., 2006).

energy. Upon reaching their critical size, the cavities would implode, release high energy, generate a high-pressure condition (up to 50 MPa), and heat surrounding to an extremely high temperature (up to 5500 °C). Since ultrasonic waves are mechanical waves, strong agitation, shear stress, and turbulence are also characterized during ultrasound processing (Yu, Seow, Ong, & Zhou, 2017). Such a unique environment generated in ultrasound is favored by the food industries as a mean of processing, cleaning, and sanitizing. Four types of ultrasound are classified according to frequency and intensity. Scientists normally define the ultrasound with a frequency higher than 500 kHz as high-frequency ultrasound, and an intensity higher than 1 W/cm2 as high-intensity ultrasound. For the high-frequency ultrasound, it is normally utilized for soft tissue surgery (using high-frequency and high-intensity focused ultrasound), or diagnostic imaging, increased drugs’ delivery, and simulation of tissue regeneration (using high-frequency and low-intensity ultrasound) (Erriu et al., 2014). The low-frequency ultrasound with either low or high intensity has been applied to the food industry as a mean of food processing and quality control. With a sufficiently low intensity of ultrasound, stable cavitation is produced without violently collapsing of bubbles during the compression cycle. When the intensity of ultrasound is high enough, cavities would implode after absorbing sufficient energy. The sudden reversal in the motion of bubble wall produces a shock wave and high temperature that can fragment water and other molecules into free radicals. Therefore, a low-frequency and high-intensity ultrasound may become a potential mean of controlling foodborne microbial biofilms in the food industry. 4. Ultrasonic or sonic treatment on foodborne microbial biofilms The effects of ultrasonic or sonic treatment on foodborne microorganisms have been reported since the 1950s, and hundreds of research papers have been published so far. Studies on ultrasonic or sonic responses of microbial biofilms are relatively new aspects which only started in recent 30 years. As shown in Table 1, these studies have indicated that the effects of ultrasound on microorganisms in biofilms may contribute to either the bactericidal effect brought by ultrasound or the stimulation of bacterial metabolism after ultrasonic or sonic treatment under certain conditions. Furthermore, these two phenomena may also co-exist when treat the microbial biofilms by sound or ultrasound.

4.2. Alteration of biofilm adhesion by ultrasound Besides the bactericidal effect in the biofilms brought by the ultrasound, it also significantly alters biofilms adhesion. The detachment of biofilm from the contact surface is one of the main purposes for adopting ultrasonic cleaning in the food industry. The effectiveness of ultrasonic cleaning again depends on the intensity of ultrasound. The mechanical oscillation generated by the low-frequency and high-intensity ultrasound is normally considered as a main reason leading to the destruction of the biofilms. Oulahal, Martial-Gros, Bonneau, and Blum (2004) have developed two ultrasonic devices, i.e. flat and curved ultrasonic transducers, for removing biofilms in milk from opened and closed surfaces, respectively. E. coli and S. aureus milk biofilms were completely removed from a stainless-steel surface in 10 s by using the flat ultrasonic transducer (40 kHz); however, the curved ultrasonic transducer at the same parameters were failed to completely remove the E. coli and S. aureus milk biofilms (30 ± 7% and 66 ± 10%, respectively). Another study also showed the effectiveness of detaching Bacillus stearothermophilus biofilm formed on a stainless-steel surface, which was 83 ± 15% after a 10 s-ultrasonic treatment at the frequency of 40 kHz (Oulahal-Lagsir, Martial-Gros, Boistier, Blum, & Bonneau, 2000). A study on ultrasonic cleaning of membrane fouling indicated that the effectiveness of dislodging biofilms was influenced by the intensity and frequency of ultrasound, as well as the distance from the ultrasonic transducer (Lim & Bai, 2003). Therefore, the effectiveness in removing foodborne microbial biofilms is varied from surface conditions, nature of food matrix, or type of microbial strains in the biofilm. Besides biofilm removal, the ultrasonication may also decrease the adhesion of microbes and inhibit their further attachments to contact surfaces as proved in a study on Streptococcus mutans. A decrease of

4.1. Bactericidal effect of ultrasound The bactericidal effect in the biofilms highly depends on parameters during the ultrasonic treatment and microbial strains. The following four studies chose L. monocytogenes biofilm as the experimental subject, and the bactericidal effect in the L. monocytogenes biofilm treated by ultrasound was varied from different conditions. Torlak and Sert (2013) evaluated the effectiveness of ultrasound at a low-frequency level (35 kHz) in eliminating L. monocytogenes biofilm formed on a polystyrene surface. Results showed that the percentage reduction of biofilm biomass was achieved to 20%, 45%, and 87% with the treatment duration of 1, 5, and 15 min, respectively. Reduction percentage of viable cells was also increased with the extended exposure time, and more than 90% of L. monocytogenes in the biofilm was inactivated after the ultrasonic treatment. Another study shows that survived L. monocytogenes 10403S biofilm formed on a stainless chip was significantly decreased treated by a power ultrasound (20 kHz, 120 W), and less than 45% of cells was survived after the 0.5 or 1.0 min-ultrasonic treatment (Baumann, Martin, & Feng, 2009). The other two studies indicated that the bactericidal effect of ultrasound was not significant. L. monocytogenes ATCC 19118 was chosen as the experimental subject, and the bactericidal effect of ultrasound (37 kHz, 1200 W for equipment) on its biofilm was not significant after a 100 min-ultrasonic treatment. The viable L. monocytogenes inoculated on stainless steel was only decreased 93

94

Polyvinyl chloride

Polyethylene

Stainless steel Stainless steel Stainless steel Polystyrene Polyurethane conveyor belts (roughness: 1.10; contact angle: 91.62) Polyvinyl chloride drain pipes Stainless steel

Cherry tomato Strawberry Fresh cucumber

Iceberg lettuce

S. aureus

P. aeruginosa

E. coli S. aureus L. monocytogenes 10403S L. monocytogenes B. cereus CCM2010

L. monocytogenes L. monocytogenes ATCC 19118

S. typhimurium ATCC 14028 S. enterica C. sakazakii

L. monocytogenes ATCC 19118

b

b

Room temperature

20 ± 4 °C 8 ± 1 °C Room temperature

Room temperature Room temperature

20 °C 20 °C 20 °C Room temperature Room temperature

N.D.

37 °C

37 °C

Temperature

s s s min min

30 s 5 min 100 min 10 min 5 min 10 min 60 min 5 min 100 min

60 60 60 15 30

2h

48 h

48 h

Duration

37 kHz, 1200 W (equipment)

45 kHz 40 kHz, 500 W 37 kHz, 380 W

30 kHz, 750 W (equipment) 37 kHz, 1200 W (equipment)

100 Hz 800 Hz 1600 Hz 100 Hz 800 Hz 1600 Hz 500 kHz, 1 mW/cm 500 kHz, 10 mW/cm 40 kHz 40 kHz 20 kHz, 750 W (equipment) 35 kHz 37 kHz, 200 W

Frequency, power/intensity of ultrasonication

Negative value of reduction percentage of viable cells refers to that the growth of microbes was simulated after sonic or ultrasonic treatment. N.D. refers to not documented.

Polyvinyl chloride

P. aeruginosa

a

Food contact surfaces

Foodborne microbial biofilms

Table 1 Effects of sonic or ultrasonic treatment alone on foodborne microbial biofilms.

3.0% 7.4% 19.1% 18.4% 10% 3.5% 18.1% 3.1% 7.7%

Murphy et al. (2016)

−45.5% −196.4% −172.7% −188.0% −532.0% −608.0% 0.3% 6.9% −1.3% 2.5% 54.8% 90% 21.7%

Na-Young et al. (2014)

José and Vanetti (2012) Rosário et al. (2017) Bang et al. (2017)

Berrang et al. (2008) Na-Young et al. (2014)

Oulahal-Lagsir et al. (2000) Oulahal-Lagsir et al. (2000) Baumann et al. (2009) Torlak and Sert (2013) Fink et al. (2017)

Qian et al. (1996)

Murphy et al. (2016)

References

Reduction percentage of viable cells a

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Trends in Food Science & Technology 96 (2020) 91–101

Trends in Food Science & Technology 96 (2020) 91–101

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Fig. 1. An example of (A) mature biofilm formed on food contact surfaces or real food surfaces; and proposed mechanisms of action and target sites on the biofilm treated by (B) chemical disinfectants alone, (C) ultrasound alone, and (D) combined ultrasound and chemical disinfectants.

biofilm may be activated, and further promote transporting nutrients and oxygen to the underlying microbes, therefore, simulating the growth of inner cells (Carmen et al., 2005; Pitt & Ross, 2003). Based on the literature reviewed in this section, it is noticeable that the either sonic or ultrasonic treatment on the mature foodborne microbial biofilms may result in totally different results under different operating conditions. The low-frequency and high-intensity ultrasound appears to have great potential as an effective method for detaching foodborne microbial biofilm from the contact surface and causing the lethal effect of microbes simultaneously. Its effectiveness depends on the duration of exposure, the distance between biofilms and the ultrasonic transducer, and types of microbial strains. As for the low-frequency and low-intensity ultrasound, it may only partially destroy some surface structures of biofilms, and most importantly, stimulate the bacterial metabolism, boost the formation of biofilm, and promote the growth of microbes simultaneously. Therefore, further verifying procedures on working parameters of ultrasound should be carefully evaluated if ultrasound is adopted alone as a mean of controlling the foodborne microbial biofilms.

bacterial adhesion was observed after a low-energy pulsed ultrasound treatment of Streptococcus mutans by an inhibitory effect on the surface protein antigen C protein and heat shock protein expression (Ishibashi et al., 2010). 4.3. Stimulation of biofilm growth by sound or ultrasound On the contrary, sonication or ultrasonication is capable of simulating microbial growth when frequency or intensity is adopted at a relatively low level. An example of P. aeruginosa exposed to a 48 h-sonic treatment with frequency ranging from 10 to 1600 Hz showed that the formation biofilm and the total number of bacteria after a sonic treatment at 800 Hz were 3 times higher than the control without the sonic treatment and slightly higher than that after a sonic treatment at 1600 Hz without significant difference. E. coli K-12 was subsequently tested with a sonic treatment at 8000 Hz and achieved the maximum biomass and growth rate of E. coli K-12 compared with the control (Gu, Zhang, & Wu, 2016). Another case of stimulating the growth of E. coli, P. aeruginosa, and Staphylococcus epidermidis inoculated on a polyethylene surface as biofilm modes was also observed after the ultrasonic treatment at 70 kHz with intensity less than 2 W/cm2 compared with those without the ultrasonic treatment; the same phenomenon was also observed at their planktonic forms (Pitt & Ross, 2003). The acoustic responses of bacteria are possibly attributed to mechanotransduction that stimulates bacterial mechanosensitive channels, e.g. MscS and MscL. The mechanical signals generated by either sonic or ultrasonic treatment may activate these mechanosensitive channels in the cells, and in turn promote the growth of cells and biofilms (Murphy et al., 2016). As consequences of sound stress at transcriptional and posttranscriptional levels, synthesis of intracellular RNA and total protein are promoted. The altered growth rate of microorganisms in the deeper layers of biofilm is another phenomenon after the sonic or ultrasonic treatment. Mature biofilms as highly organized ecosystems disperse several channels which provide passages for exchanging water, metabolites, nutrients, and waste products with the outer environment (Sauer, Rickard, & Davies, 2007). Resistances of microbes in the deep layers of thick aggregates of biofilm are attributed to slow microbial metabolisms due to low oxygen content and insufficient nutrients (Hassan, Anand, & Avadhanula, 2010). The sonic or ultrasonic treatment with a low-intensity level may only peel off the surface structure of biofilm and lead to a limited bactericidal effect. The inner channels in the

5. Combined treatment of ultrasonication and chemical disinfectants against foodborne microbial biofilms Many studies have revealed the combined effects of ultrasound and antibacterial agents on biofilms control in the domain of medicine. In this section, we have reviewed recent studies focused on how to control foodborne microbial biofilms in the food industry with combined treatment of ultrasound and chemical disinfectants. As mentioned previously, the bactericidal effect of foodborne microbial biofilm after the ultrasonic treatment normally cannot achieve desired results (Qian, Stoodley, & Pitt, 1996). The resistance of microbial biofilms to chemical disinfectants and antibiotics is also commonly reported as comprehensively reviewed in (Bridier et al., 2011; Stewart & William Costerton, 2001). With the development of ultrasound technology, the combination of ultrasound and chemical disinfectants as an emerging strategy has gradually adopted to effectively control foodborne microbial biofilms, as shown in Table 2. Three disinfectants, i.e. hydrogen peroxide (111 ppm), quaternary ammonium compounds (400 ppm), and chlorine-based sanitizer (600 ppm), were failed to effectively inactive L. monocytogenes biofilm enumerated inside a wall surface of polyvinyl chloride drain pipe. With the assistant of ultrasonication (30 kHz, 750 W), it improved the 95

Polystyrene

Polyurethane (conveyor belts with roughness of 1.10, and contact angle of 91.62) Polyvinyl chloride (drain pipes)

Stainless steel

L. monocytogenes

B. cereus CCM2010

L. monocytogenes ATCC 19118

96

Strawberry

Fresh cucumber

Iceberg lettuce

C. sakazakii

L. monocytogenes ATCC 19118

Room temperature

Room temperature

8 ± 1 °C

20 ± 4 °C

Room temperature

Room temperature

Room temperature Room temperature

Temperature

Sodium hypochlorite

Acetic acid Sodium dodecylbenzenesulfonate Peracetic acid Peroxyacetic acid

Peracetic acid

Hydrogen peroxideperoxyacetic acid Quaternary ammonium Chlorine Sodium hypochlorite

P3-Tresolin ST®

Benzalkonium chloride

Chemical disinfectants

500 mg/L 1200 mg/L 40 mg/L 50 ppm 100 ppm 150 ppm 200 ppm 50 ppm 100 ppm 150 ppm 200 ppm 200 ppm 200 ppm

5 min 10 min 10 min 10 min 10 min 60 min 60 min 60 min 60 min 5 min 100 min

40 mg/L

5 min 5 min

10 min

400 600 200 200

30 s 30 s 5 min 100 min

ppm ppm ppm ppm

111 + 473.6 ppm

100 mg/L 400 mg/L 10% alcohols, 2.5% benzakolium chloride, 2.5% didecyldimethylammonium chloride

Concentration

30 s

15 min 15 min 30 min

Duration

100% 15.3 27.1 38.9 45.1 32.6 47.2 52.8 64.6 68.0% 84.6%

5.0% 1.7%

86.2%

69.8% 56.9% 66.2% 85.3%

52.8%

93.1% 99.4% 60.9%

Reduction percentage of viable cells

0.91 1.22 1.18 1.10 0.97 1.21 1.26 1.06 1.06 1.02 1.20

0.20 0.14

1.09

1.04 1.58 1.02 1.12

1.34

0.77 0.66 1.17

Synergistic values a

Na-Young et al. (2014)

Bang et al. (2017)

José and Vanetti (2012) Rosário et al. (2017)

Na-Young et al. (2014)

Berrang et al. (2008)

Torlak and Sert (2013) Fink et al. (2017)

References

a Synergistic value = Reduction percentage of viable cells after combined treatment of ultrasonication and chemical disinfectant/(reduction percentage of viable cells treated by ultrasonication alone + reduction percentage of viable cells treated by chemical disinfectant alone). Synergistic values in Table 2 are derived from raw data reported in the listed references.

37 kHz, 1200 W (power for equipment)

37 kHz, 380 W

40 kHz, 500 W

Cherry tomato

S. typhimurium ATCC 14028 S. enterica

30 kHz, 750 W (power for equipment)

37 kHz, 200 W

35 kHz

Frequency, power/intensity of ultrasound

37 kHz, 1200 W (power for equipment) 45 kHz

L. monocytogenes

Food contact surfaces or real food surfaces

Foodborne microbial biofilm

Table 2 Combined treatment of ultrasound and chemical disinfectants against foodborne microbial biofilms.

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the highest reduction of S. typhimurium biofilm was achieved from 7.7 to 0.4 log10 CFU/g after the same treatment. Moreover, the sanitized cherry tomatoes did not observe any damage of surfaces after the 10 min-ultrasonication. Another study chose strawberry as the subject and subsequently evaluated the effectiveness of Salmonella enterica biofilm inactivation and corresponding alteration regarding physicochemical and sensory of sanitized strawberry during storage (Rosário et al., 2017). A 5 mincombined treatment of ultrasonication (40 kHz, 500 W) and peracetic acid (40 mg/L) showed the best performance, which reduced 2.1 log10 CFU/g of viable cells during the storage (9 days). However, acetic acid (500 mg/L) and sodium dodecylbenzenesulfonate (1200 mg/L) treatments were not significantly increased by the ultrasound due to low efficiency of the two chemicals in the inactivation of S. enterica. Therefore, it is again proved that the bactericidal effect is mainly attributed to the chemical disinfectants instead of ultrasound. Moreover, it should be very cautious when choosing suitable sanitizer for strawberry since color difference became detectable after treatments of sodium hypochlorite (200 μg/mL) and hydrogen peroxide (5%) for 2 min (Alexandre, Brandão, & Silva, 2012). As another benefit of the ultrasonication, it is possible that the enzymes leading to browning were inactived in the strawberries; therefore, no significant change of color during the 9 d-storage was observed after the ultrasonic processing. In addition, there was no significant alteration of firmness (taken pectin as an indicator) in the sanitized strawberry after the combined treatments. Two fresh vegetables were subjected to combined treatment of ultrasonication and chemical disinfectants and achieved a synergetic effect of removing foodborne microbial biofilms. For decontamination of Cronobacter sakazakii ATCC 12868 biofilm formed on fresh cucumbers, the reduction of viable cells was 3.51 log10 CFU/cm2 after the combined treatment of ultrasonication (37 kHz, 380 W) and 200 ppm peroxyacetic acid for 60 min. Furthermore, hunter color, moisture contents, hardness and chewiness of sanitized cucumber were not significantly altered after the treatment (Bang, Park, Kim, Rahaman, & Ha, 2017). A similar study on controlling L. monocytogenes biofilm on iceberg lettuce indicated that the combined treatment of ultrasonication (37 kHz, 380 W) and sodium hypochlorite (200 ppm) resulted in the highest bacterial reduction (6.51 log10 CFU/g), which was greater than either ultrasonication (0.68 log10 CFU/g) or treatment of sodium hypochlorite alone (4.17 log10 CFU/g) (Na-Young et al., 2014). The chemical resistance of biofilms is mainly attributed to the limited diffusion and reaction of chemical disinfectants in biofilms (as shown in Fig. 1B) due to a compact and complex eco-system established by multiple layers of inner cells and EPS structure (Gutierrez-Pacheco et al., 2018). The EPS is composed of polysaccharides as fundamental components and other constituents, including proteins, lipids, nucleic acids, etc. (Donlan, 2002). Most types of EPS in the biofilm are both hydrophobic and hydrophilic, and these characters allow the EPS structure to give full protection of inner microbes against hostile environments (e.g. dehydration) as well as penetration and diffusion of chemical disinfectants through binding them directly (Shi & Zhu, 2009). For now, almost all the studies elucidated the destruction of the biofilm structure as a consequence of mechanical oscillation during the ultrasonic treatment. The mechanical oscillation generated by ultrasound has a capacity to remove microbial biofilm, expose inner cells, and aid the disinfectants in reaching target spots, e.g. slots and cracks on fresh foods surface for increasing the effectiveness of chemical treatment on the biofilms (Sagong et al., 2013). However, there is no study that fully recognizes whether sono-degradation of EPS exists during the treatment of ultrasound. Protein concentration and fluorescence matters in the EPS were removed by ozone as a strong oxide through attacking specific amino acid residues on the protein chain as reported by Meng, Xi, and Yeung (2016). It is reasonable to speculate that some oxides generated during the ultrasonication may also play the same role in inactivating extracellular proteins in the EPS. Microbes in the deeper layers of mature biofilms have very low

effectiveness of chlorine-based sanitizer (increased 45.22% of cell reduction) and quaternary ammonium compounds (increased 72.15% of cell reduction) against the L. monocytogenes biofilm formed on the same drain pipe. The authors also counted viable cell numbers in diluent after the ultrasonication and pointed out that the ultrasonication would not release more cells into a planktonic state. From our viewpoint, such a conclusion may not be solid since the bactericidal effect of ultrasonic treatment would be stronger when targeted the planktonic cells; meanwhile, the detection method adopted in this study did not measure the number of dead cells in the diluent. Furthermore, the ultrasonic treatment is regarded as an all-or-nothing process as concluded in Li et al. (2016). Therefore, the antibacterial action of ultrasound would be irreversible. Moreover, it is interesting to notice that the effectiveness of peroxyacetic acid as an oxidizing sanitizer was not significantly promoted even with the addition of ultrasonic treatment (Berrang et al., 2008). It is again proved that the bactericidal effect of ultrasonication is very limited, but is mainly attributed to the chemical disinfectants when treated the biofilms. Another study chose Bacillus cereus biofilm as an experimental subject isolated from polyurethane conveyor belts in bakeries. The biofilm inoculated from B. cereus spores appeared to be more firmly attached to the polyurethane surface compared with that from vegetative cells, which may be attributed to different proteins on the B. cereus exosporium (Soni, Oey, Silcock, & Bremer, 2016). Using either 30 min-ultrasonication (37 kHz, 200 W) or 2% P3-Tresolin ST® (10% alcohols, 2.5% benzakolium chloride, 2.5% didecyldimethylammonium chloride) as a commercial cleaning agent alone did not successfully remove the biofilm and eliminate the cells. The highest cleaning efficacy of B. cereus biofilm was achieved with the combination of ultrasonication and 2% P3-Tresolin ST®. Taken the conveyor belts as an example in the bakery industry, the authors suggested to always include hygiene management for food contact surface through implementing hurdle technologies for achieving synergetic effects (Fink, Oder, Stražar, & Filip, 2017). Polystyrene with high hydrophobicity is a commonly used food contact surface in the food industry, e.g. conveyer belts, cutting board, gaskets, etc. Another study has revealed the resistance of L. monocytogenes biofilm formed on polystyrene to benzalkonium chloride (400 mg/L), which only reduced 60% of viable cells after 15 min. Furthermore, an ultrasonic treatment (35 kHz) combined with benzalkonium chloride at either 100 or 400 mg/L was achieved higher than 90% or 95% reduction of viable cells (Torlak & Sert, 2013). Moreover, the authors suggested to conduct furthering studies on evaluating the effectiveness of controlling L. monocytogenes biofilm formed on hydrophobic contact surfaces instead of hydrophilic one since a higher number of cells preferred to adhere to a food contact surface with high hydrophobicity (Sinde & Carballo, 2000). Decontamination of foodborne microbial biofilm is a critical procedure for producing high-quality fresh foods. The following two studies have adopted the ultrasound as an auxiliary technology during sanitizing fresh fruits without compromising their qualities, e.g. cherry tomato, and strawberry. José and Vanetti (2012) evaluated the effectiveness of combined ultrasonication (45 kHz) and four disinfectants, i.e. sodium dichloroisocyanurate (20 and 200 mg/L), hydrogen peroxide (5%), chlorine dioxide (10 mg/L), and peracetic acid (40 mg/L), during the decontamination of natural contaminant microbiota and S. typhimurium ATCC 14028 biofilm on minimally processed cherry tomatoes. The natural contaminant microbiota was classified as two groups, i.e. aerobic mesophiles as well as molds and yeast. The combined treatment of ultrasound and chemical disinfectants on the cherry tomatoes was achieved a higher reduction log of tested microbes compared with that treated by a single method (either ultrasonication or chemical disinfectants) at the same conditions. The highest reductions of aerobic mesophiles (4.4 log10 CFU/g) and the group of molds and yeast (3.4 log10 CFU/g) were achieved with a 10 min-combined treatment of ultrasonication and 40 mg/L peracetic acid; meanwhile, 97

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proportion of divalent cations. As mentioned previously, polysaccharides are primary components in the EPS as fundamental structures of the biofilm. For gram-positive microbial biofilm, some polysaccharides are neutral or polyanionic in the EPS that allows the association of divalent cations, e.g. Mg2+ and Ca2+; the constituents of EPS in gram-negative microbial biofilm may be primarily cationic (Donlan, 2002). A positive correlation between the concentration of calcium and the accumulated amount of biofilm has been indicated in many studies. The chelating agents may weaken the matrix of biofilm by screening out crosslinking electrostatic interactions (Chen & Stewart, 2000). Taking EDTA as an example, it is capable of disrupting the outer cell envelope by chelating Mg2+ and Ca2+ as stabilizers of the lipopolysaccharide layer in Gram-negative microbes or peptidoglycan layer in Gram-positive microbes, therefore, leading to the biofilm removal (Stevens, Klapes, Sheldon, & Klaenhammer, 1992). Martial-Gros, Bonneau, and Blum (2004) developed a curved ultrasonic transducer to remove biofilms from closed surfaces. The established E. coli and S. aureus biofilms on stainless-steel were to simulate real scenarios in the dairy industry. Combined the ultrasonication with either EDTA or ethylene glycol-bis-(β-aminoethyl ether)-N,N-tetraacetic acid (EGTA) solution at a concentration of 0.05 mol/L, it was completely removed the E. coli biofilm, which was significantly higher than that treated by the ultrasonication alone (percentage of biofilm removal = 30%). However, there was no synergistic effect between the ultrasonication and the two chelating agents when treated with the S. aureus biofilm; and the percentages of biofilm removal were determined to be 74% and 47% for EDTA and EGTA, respectively. The authors also conducted a similar study on the same bacterial biofilms isolated from meat; however, inconsistent results were obtained. The combined ultrasonication (40 kHz, 10 s) and EDTA (0.025 mol/L) treatment significantly enhanced the percentage of S. aureus biofilm removal (89 ± 8%) compared with that treated by the ultrasonication alone (39 ± 5%). For removing the E. coli biofilm, in turn, the synergistic effect was limited, which only increased the percentage of biofilm removal from 49 ± 5% to 58 ± 8%. The authors emphasized that both the ultrasonication and the two chelating agents adopted in this study did not cause detectable killing in both E. coli or S. aureus (Oulahal & Martial, 2007). Both studies have indicated mechanisms of biofilm removal by either ultrasonication or chelating agents, but insufficient mechanisms of synergistic effect were proposed. In addition, no further explanations are given since the inconsistent findings of biofilm removal efficiency of E. coli as a Gram-negative bacterium and S. aureus as a Gram-positive bacterium.

activations, which is comparable to spore with the high resistance of chemical disinfectants (as shown in Fig. 1A with dark green color). These cells in the deeper layers of mature biofilms are stimulated by the ultrasound that makes them more sensitive to chemical disinfectants (as shown in Fig. 1D). The promoted transportation of oxygen and nutrients to the attached cell would allow an increase of chemical penetration and promote the bactericidal effect of inner cells. Besides the poor penetration of chemical disinfectants in the biofilms, many food items have folded leaves and hydrophobic pockets make the disinfectants not effective (Babic, Roy, Watada, & Wergin, 1996). Therefore, the ultrasonication would become a suitable auxiliary technology combined with the chemical treatment for decontamination soils and biofilms formed on either food contact surfaces or real foods. 6. Combined treatment of ultrasonication and other strategies against foodborne microbial biofilms Besides chemical disinfectants combined with ultrasound as a mean of controlling foodborne microbial biofilms, ozone or electrolyzed water combined with ultrasound are innovative strategies for inactivating foodborne microbial biofilms. In addition, chelating agents and enzymes are also reported as innovative strategies combined with ultrasound for removing biofilms instead of achieving a strong bactericidal effect. 6.1. Ozone and electrolyzed water Ozone would effectively remove protein concentration and fluorescence matters in the EPS through attacking specific amino acid residues on the protein chain instead of polysaccharides degradation (Meng et al., 2016). Baumann et al. (2009) reported the biofilm removal and bactericidal effect of L. monocytogenes biofilm formed on stainless steel by using combined treatment with ultrasonication and ozone. The ozone was cycled through a potassium phosphate butter that achieved different concentrations of ozone in the solution ranging from 0.25 to 1.00 ppm. Utilization of either ultrasonication (20 kHz, 60 s) or ozonation (0.50 ppm, 60 s) alone was only reduced the viable L. monocytogenes to 3.35 and 4.95 log10 CFU/mL, respectively. The combined treatments of the ultrasonication and either 0.50 ppm ozone for 60 s or 1.00 ppm ozone for 30 s were able to eliminate the viable L. monocytogenes in the biofilm. Electrolyzed water is normally obtained by the electrolysis of a dilute sodium chloride solution. The electrolyzed water is gaining increasing popularity because of a strong sanitizing effect and easy production without polluting the environment (Yang, Feirtag, & DiezGonzalez, 2013). The combined treatment of ultrasonication (37 kHz, 80 W) and electrolyzed water (pH = 7.0, free available chlorine = 4 mg/L) was first adopted to inactive E. coli, Pichia pastoris, and Aureobasidium pullulans biofilms formed on stainless-steel coupons as reported in Zhao, Zhang, and Yang (2017). The combined treatment significantly reduced the survival cells both on the stainless-steel coupons and in suspension for the three strains. The main function of ultrasonication, therefore, is the detachment of biofilm and release the cells into the suspension. The free available chlorine component in the electrolyzed water would attack the cells more effectively resulting in more sanitizing effect both for cells in the biofilm and the suspension. The combined treatments of ultrasonication and ozonation or electrolyzed water have great potential as promising approaches for sanitizing food equipment and real food surfaces.

6.3. Enzymes Enzymes have high specific and rapid reaction rates under mild conditions, which make them suitable for detaching biofilm from contact surfaces through degrading matrix polymers of biofilm directly. Five enzymes, i.e. protease type XXIII (36 U/mL), crystalline trypsin (7600 U/mL), amyloglucosidase (50 U/mL), papain (3 U/mL), and lysozyme (6000 U/mL), were tested combined with ultrasonication (40 kHz, 10 s) and EDTA solution (0.025 mol/L), and further evaluated their performances on removing E. coli and S. aureus biofilms. The highest percentage of E. coli biofilm removal was achieved in the combined treatment with protease and papin (75 ± 4%), and a moderate removal percentage was achieved in the combined treatment with amyloglucosidase (42 ± 1%). Moreover, the same treatment with amyloglucosidase was capable of completely removing the S. aureus biofilm, and the combined treatment with trypsin and lysozyme was also achieved the same result. Therefore, as concluded by the authors, the removal of E. coli biofilm was favored by proteolytic enzymes that showed a synergistic action, and amyloglucosidase was mainly acted on polysaccharides embedding biofilm cells. Facing to the heterogeneity of proteins and extracellular polysaccharides in the biofilm substances, applications of different enzymes specificities are recommended for

6.2. Chelating agents Aminopolycarboxylic acids, primarily ethylene–diamine tetraacetic acid (EDTA), are counted for more than 70% of chelating agents consumed in the world. Chelating agents’ solution with metal ions is commonly used to remove inorganic scales that contain a high 98

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after combing with other strategies. It is noticeable that the bactericidal effect of many chemical disinfectants in the biofilms is also very limited since the EPS structure to give full protection of inner microbes against the penetration and diffusion of chemical disinfectants through the EPS as a physical barrier or binding the chemicals directly. Therefore, the combined treatment of ultrasonication and chemical disinfectants becomes one of the most practical strategies for controlling foodborne microbial biofilms. To verify the synergistic effect between ultrasonication and chemical disinfectants, synergistic values were derived from raw data reported in the references, as shown in Table 2. For most of studies, the synergistic values are higher than 1.00 which proves the existence of the synergistic effect. Moreover, the reduction percentage of viable cells was achieved higher than 80% after adopting the combined treatment as reported in many studies. Types and concentration of chemical disinfectants selected in these studies are also suitable to the food industry that makes the combined treatments as potential strategies for controlling the foodborne microbial biofilms. The following factors of the combined treatments may significantly influence the effectiveness of biofilm removal and inactivation of microorganisms in the biofilms:

sufficiently degrading the biofilms (Oulahal & Martial, 2007). Although studies on the combined treatment of ultrasonication and chelating agents/enzymes are still limited currently, it would become a potentially emerging strategy for rapidly removing biofilms, flushing the detached biofilms with flowing liquid, and eventually achieving the biofilm control. 7. Discussion and conclusions The application of ultrasound technology on controlling foodborne microbial biofilms has been gradually developing in recent years. Either sonication or ultrasonication has further incorporated with chemical disinfectants, chelating agents, enzymes, and other strategies for achieving a reasonably high bactericidal effect and biofilms removal from food contact surfaces. During the ultrasonication, frequency and intensity of ultrasound are the two determining factors. The low-frequency and high-intensity ultrasound would contribute to the bactericidal effect in the biofilms and biofilms removal; however, the low-frequency and low-intensity ultrasound would stimulate the growth of microbes in the biofilms. As summarized in Table 1, the treatment of either sonication with the frequency lower than 20 kHz or ultrasonication with intensity lower than 1 W/cm2 would stimulate the growth of microbes. Possible mechanisms are concluded as follows:

1) Microbial strains and growing stages of biofilms; 2) Types and concentration of chemical disinfectants; 3) Working parameters of ultrasonication, e.g. frequency and power/ intensity; 4) Other conditions, e.g. time, temperature, and food contact surfaces (roughness, contact angle, etc.).

1) Mechanotransduction generated by the sonication or ultrasonication would stimulate bacterial mechanosensitive channels which contribute to promoting the synthesis of intracellular RNA and total protein; 2) Channels for exchanging metabolites, nutrients, and waste products would be activated, and subsequently increase the growth rate of microorganisms in the deeper layers of biofilms.

Besides the above-mentioned chemical agents, ozone and electrolyzed water are the two emerging strategies for inactivating microorganisms. Ozone itself would effectively remove protein concentration and fluorescence matters in the EPS through attacking specific amino acid residues on the protein chain instead of polysaccharides degradation. Meanwhile, bactericidal mechanisms of ozone are concluded as the increase of cytoplasmic membrane permeability and cytoplasm coagulation. Electrolyzed water can be classified as strong acid electrolyzed water and low concentration electrolyzed water. The potential application of the strong acid electrolyzed water is limited due to corrosive characteristic (pH ≤ 2.7). The low concentration electrolyzed water is newly developed with a relatively high pH of 6.2–6.5 and a concentration of free chlorine (almost all hypochlorous acid) about 2–5 mg/L. The hypochlorous acid (HOCl) is the most effective form of chlorine compounds that would inhibit glucose oxidation by chlorineoxidizing sulfhydryl groups of certain enzymes important in carbohydrate metabolism (Rahman, Ding, & Oh, 2010). After combining with ultrasound, the bactericidal effect was significantly promoted compared with those adopted one strategy alone since the ultrasonication would promote permeability of ozone or electrolyzed water in the biofilms, partially disrupt biofilm structure and cell membrane, and eventually lead to cell lysis. The effectiveness of the combined treatments is influenced by the same factors as concluded above. Chelating agents and enzymes are the two innovative strategies combined with ultrasound for effectively removing biofilms instead of achieving a strong bactericidal effect. Chelating agents can weaken the matrix of biofilm by screening out crosslinking electrostatic interactions. The enzymatic treatment can degrade matrix polymers in the biofilms. Therefore, both treatments would achieve detaching biofilms from food contact surfaces. For now, only a few studies adopted the combined ultrasonication and chelating agents or enzymes on the biofilm removal, and all of them have reported a promoted effectiveness of biofilm removal compared with that adopted one strategy alone.

The low-frequency and high-intensity ultrasound is capable of contributing to the bactericidal effect in foodborne microbial biofilms; however, such a bactericidal effect is very limited as shown in Table 1. For most of studies, the reduction percentage of viable cells was less than 20% in the foodborne microbial biofilms. Although the extended duration and/or increased intensity of ultrasound would contribute to a better bactericidal effect, such an enhancement is still very limited. These two ways are practical in the food industry due to the limitation of treatment time (usually a few minutes), high consumption of energy, and high cost of powerful ultrasonic generators. Mechanisms of lowfrequency and high-intensity ultrasound on bactericidal effect and biofilm removal include: 1) Mechanical oscillation as a physical mechanism would partially destroy the fundamental structure of biofilms and change the integrity of cells, which result in biofilms detachment and lethal effect of microbes, respectively; 2) An extremely high, albeit momentary temperature and pressure environment generated by ultrasound would trigger a localized heating and further inactive microbes in the biofilms; 3) Water and other molecules are fragmented into free radicals and further generate strong oxides as bactericides. These oxides may attack specific amino acid residues on the protein chain in the EPS and cell membrane, and further alter the permeability of biofilms and cell membrane. As consequences of cavities generated by the low-frequency and high-intensity ultrasound, degradation matrix polymers of biofilm, change of biofilm and cell membrane permeability, as well as lethal effect of foodborne microorganisms, may happen simultaneously. Since the bactericidal effect brought by the ultrasonication itself is relatively limited, many positive aspects brought by the ultrasonication make it more practical on controlling foodborne microbial biofilms

8. Recommendations for future aspects Frequency and power (or intensity) of ultrasound are the two determining factors for biofilm removal and bactericidal effect. We 99

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References

noticed that more than half of the research papers reviewed in this article did not report the both values. The output power (not the power of ultrasonic equipment) or the intensity of ultrasound was normally absent in many studies. Therefore, it is strongly recommended to report both parameters of ultrasound simultaneously in the future publication. In addition, acoustic energy consumed during the ultrasonication is better to be reported as well. This value can be easily measured by calorimetry which is normally connected to the ultrasonic generator (Koda, Kimura, Kondo, & Mitome, 2003). Multi-physics modeling is another promising method for simulating distributions of acoustic pressure and temperature during ultrasonic processing (Yu, Gao, et al., 2018). Such a simulation would provide additional information about acoustic pressure and temperature distribution on the surfaces of biofilms during the ultrasonication. In addition, the synergistic value is strongly recommended to be reported as a straightforward indicator of synergistic effect when the combined treatment is adopted. Many investigations from genetic aspects have been conducted for revealing biofilm-related gene expression under various environmental stresses, such as cold, heat, acid, oxidative, high-pressure stresses, etc. (De Angelis & Gobbetti, 2011). There are still uncertainties on changes of foodborne microbial biofilm under a “sonic or ultrasonic stress”. Since the stimulation of microbial metabolisms after certain sonic or ultrasonic treatments has been observed as a unique phenomenon, it would be more meaningful to further reveal related gene modulation and expression in the biofilms. QS mechanisms are also recommended to be revealed, which would give a better understanding of ultrasonic actions. It is glad to know that many scholars in the world are dedicating to study ultrasound-involved emerging strategies for controlling foodborne microbial biofilms in the food industry. Some papers reviewed in this article focused on controlling the biofilms formed on food contact surfaces, such as polyvinyl chloride, polyethylene, polystyrene, and stainless steel. And all the food contact surfaces commonly appear in the food industry as materials for packaging, belts, pipes, processors, and containers. These ultrasound-involved emerging strategies would have great potentials to further develop as cleaning and disinfecting methods in the food industry. Some other studies aimed to resolve the biofilm pollution which was appeared on the surfaces of fresh foods, e.g. strawberry, cucumber, lettuce, and cherry tomato. It is also worthy to further study how to effectively decontaminate the biofilms through the ultrasound-involved emerging strategies for extending shelf-life without compromising the quality of fresh foods. Besides the two research areas mentioned above, ultrasound may also potentially combine with other technologies for achieving the biofilm control, e.g. heating, high pressure, irradiation, etc. Meanwhile, more fundamental studies on elucidating mechanisms of ultrasound on foodborne microbial biofilms control are recommended as well.

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Declaration of competing interest All authors declare no competing interests. The manuscript was prepared by H. Yu. Preparation of figures and tables were conducted by H. Yu, Y. Liu, and L. Li. The overall study concept was designed by H. Yu, Y. Xie, Y. Cheng, and W. Yao.

Acknowledgments The following funding sources are gratefully acknowledged: National Key R&D Program of China (2018YFC1602300), China Postdoctoral Science Foundation funded project (2018M642165), the Fundamental Research Funds for the Central Universities (JUSRP11904), the Natural Science Foundation of Jiangsu Province (BK20171139), and Yangtze River Delta Project of Shanghai (18395810200). 100

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