Strategies,for,controlling,biofilm,formation,in,food,industry

Tingwei Zhu*,Chenxin Yng*,Xuerui Bo,Fusheng ChenXingfeng Guo

a College of Food Science and Engineering,Henan University of Technology,Zhengzhou 450001,China

b Faculty of Pharmaceutical Sciences,Gent University,Gent B9000,Belgium

Keywords:Biofilm Control strategies Essential oil Enzymes Biosurfactants

A B S T R A C T Foodborne pathogen poses a threat to the food industries as many outbreaks have been found to be associated with biofilm formation.The formation of biofilm is a self-protection growth pattern of bacteria,which increases post-processing contamination and risk to public health.It is difficult to eliminate the biofilm in the food industries,since the biofilm cells have a barrier preventing or lessening the contact with environmental stresses,antimicrobial agents and the host immune system.Bacterial biofilm formation is a complex process,including initial attachment stage,irreversible attachment stage,biofilm development stage,biofilm maturation stage,and biofilm dispersion stage.The genetic mechanism,substratum and bacterial cell surface properties involve in the biofilm formation.The biofilm inhibition methods studied are physical treatment,chemical and biochemical treatment.The potential green and safe biochemical method attracts more attention,especially,the novel strategies using the safe biochemical agents(essential oils,enzymes,biosurfactants,others)constantly emerged.The review emphasized on effective strategies for inhibiting biofilm formation in different stages(initial irreversible attachment,formation,and maturation)by use of biochemical agents,aiming to provide new insight into biofilm control in food industry thus improving food quality and safety.

Foodborne bacteria consumed along with food cover a wide range of public health concerns worldwide[1].About 600 million people globally are affected by foodborne diseases such as vomiting,diarrhea,enteritis,abdominal pain,headache,which have become a serious problem in a wide range of food industries,including meat processing,brewing,dairy and poultry[2].Around more than half of persistent infections are caused by bacterial biofilms in the United States[1].The formation of biofilm indicates a complex bacterial lifestyle that provides protection from environmental stress.An estimated 80% of all bacterial infections are biofilm-related.The foodborne pathogens includingSalmonellasp.,Listeria monocytogenes,Campylobacter jejuni,Escherichia coliO157:H7,Yersinia enterocoliticaandStaphylococcusspp.were reported on biofilm formation in food processing plants[3].

Bacteria in nature and food systems are most often attached to solid surfaces,which are helpful for their viability and growth.Furthermore,pathogenic bacteria accumulate in different equipment surfaces and finally form biofilms[4].Bacterial biofilm is the most common microbial formation;more than 90%of bacteria exist in the form of biofilms and develop biofilms on biotic and abiotic surfaces,such as stainless steel,rubber gloves,plastic,silicon rubber,and glass,used throughout the food industry during food processing[5].Biofilm-related microbial pollution poses a serious threat to food quality and safety,similar to the metal corrosion of the pipelines and tanks,causing the reduction of heat-transfer efficacy,and the deterioration of food quality and shortening of shelf-life,thus adversely affecting the economic activity of food industry enterprises because of food spoilage and equipment erosion[6].Compared with planktonic cells,biofilms have significantly increased resistance to antimicrobials,therefore,foodborne bacteria can easily survive under commonly encountered stresses when the biofilm is formed.Bacterial biofilms were first discovered on the tooth surface in which an adherent colony was formed[7].Then the phenomenon was also found in the dairy products and produce,such as soft cheeses,celery,sprouts,cantaloupe,and ice cream[8].With the development of new techniques,including scanning electron microscopy(SEM)and confocal laser scanning microscopy(CLSM),research into biochemical biofilms has gradually expanded and deepened.The structure of biofilm architecture ofL.monocytogeneson rubber gloves and plastic surfaces were depicted by the field-emission scanning electron microscopy(FE-SEM)images,and a biofilm of densely interconnected live cells was observed from the CLSM images[9].Currently biofilms are identified as surface-attached,microbially-derived sessile communities,with three-dimensional structures made up of secreted extracellular polymeric substances(EPS)[10,11].At first,the adherent cells were overlaid by a so-called conditioning film comprising a small quantity of macromolecules,such as EPS consisting of extracellular polysaccharides,proteins,phospholipids and even DNA[12].With the changes of material surface properties and environmental parameters such as the pH,nutrient levels and temperature,the adherent bacteria continue produce a polysaccharide to facilitate microcolony formation and biofilm maturation.During the maturation process,EPS helps strengthen the bond between the bacteria and the substratum,and the cell-to-cell communication(quorum sensing)embraces and stabilizes the colony[1].From a molecular view,polysaccharide intracellular adhesion(PIA)factor encoded by specific genes caused intracellular adhesion and protected bacterias from immune response and hostile environment[12].

Bacterial biofilms are common in processed food,including dairy[13],processed seafood[14],meat,and ready-toeat foods[2,15].The formation of pathogenic biofilm increases food safety risk since it has a higher ability to survive under stringent conditions[16].Therefore,analytical methods for microbial biofilm formation are helpful for new elimination strategies for bacterial biofilms in the food-processing industry,while it is important to prevent and control biofilm formation in food facilities.The conventional control strategies for cleaning and disinfection methods used in food processing plants,such as physical based-methods(clean-in-place)and chemical-based methods(using sodium hypochlorite,hydrogen peroxide,ozone,and peracetic acid),increased recurrent contamination risks and rapid deterioration of food by biofilm cells[17].Hence,researching methods and strategies for effectively combating bacterial biofilm formation and eradicating mature biofilms is essential,and promising approaches to control bacteria and their biofilms(include initial irreversible attachment stage,formation stage,and maturation stage)are emerging in recent years.The objective of the review is to summarize the efficacy of strategies for controlling biofilm formation in food industry by using biochemical agents based on natural ingredients and provide an overview of recent promising biofilm control technologies in the food processing sector.

Bacterial biofilm formation is a series of dynamic steps,comprised of initial attachment,irreversible attachment,biofilm development,biofilm maturation,and biofilm dispersion.First,bacteria in an inhospitable environment(such as pH,high temperature and salt,strong alkali and acid,excessive ultraviolet,concentration,utrients,and overuse of antibiotics,detergents,or sanitizers),actively or passively attach onto the surface of the transportation or food.The initial attachment is reversible,and only a small quantity of EPS is produced[18].During this step,the bacterial cell easily detaches and recovers the original floating lifestyle.Contact forces in this stage are mainly van der Waals force,electrostatic forces,and Lewis acidbase interaction forces[19-21].Second,with more EPS produced,the adhesion of the bacterial changes from reversible to irreversible.During this step,the contact forces transform from weak cell-cell interaction to tight bonding with the appearance of more EPS[18,22].Third,in the initial stage of biofilm development,the bacterial cells accumulate and form a microcolony,in which EPS helps stabilize the colony under any environmental stress[2].In addition,cell-cell signaling molecules are produced.The bacterial biofilm then gradually matures,and the EPS are transported to and within the biofilm.During biofilm maturation,the bacteria develop into an organized flat or mushroom-shaped structure.During the whole bacterial biofilm formation,the final step is a dispersion of the biofilm,in which the bacteria revert to the planktonic form[2].The attached material properties and environment(pH,temperature,and nutrients)can easily affect the formation and development of the biofilm[18,23,24].Thus,it is indispensable to develop effective methods to control and eliminate the formation of biofilms.

In the food industry,both physical and chemical approaches have been studied to inhibit bacterial biofilms.Previous studies have proven that mechanical treatment such as clean-in-place cannot remove all of the bacterial cells,while chemical treatment has some contributions to eliminate bacterial cells and inhibit biofilm formation[25].It was found that sodium hypochlorite(NaClO)acts as a potential disinfectant capable of inhibiting bacteria,while,the bacteria proliferation may happen under higher concentrations of disinfectants which created conditions for the biofilm formation[11].To combat foodborne infections resulting from biofilms,various methods have been developed from different aspects,such as inhibition of bacterial adhesion and bacterial detection in the early stage.Additionally,dissolving the biofilm and transforming the sessile bacteria into sensitive planktonic cells are essential[15].A lot of literature data are devoted to the study of the antimicrobial agents and inhibitory effects of biological agents and novel strategies on biofilm formation[26].Therein,the biochemical agents including essential oils(EOs),enzymes,biosurfactants,and others have been discussed on the basis of safe and“green”approaches to control pathogen biofilms formation(Fig.1).

3.1.Essential oils(EOs)

Generally,the natural and non-toxic antimicrobial agents are preferred in order to meet the health and ecological concerns.Essential oils(EOs),which is a kind of plant-derived secondary metabolites,is known as natural broad-spectrum antimicrobials that exhibit certain bacteriostatic activity for various foodborne pathogens[27].The main active ingredients in EOs are terpenes,oxygenated derivatives,and terpenoids(aromatic compounds,aliphatic acid esters,phenolic compounds).Among the derivatives,the compound phenol is considered to be the most antimicrobial active,followed by aldehydes,ketones,alcohols,ethers,and hydrocarbons[28].With the deepening research on the antimicrobial activity of EOs,study also has found that some EOs have certain effect on the biofilm formation[29,30].Murraya koenigiiEOs have been proven to reduce the formation ofPseudomonas aeruginosaPAO1 biofilm by 80%;this is accompanied by the decrease in EPS,swarming motility,and rhamnolipid production[29].Vázquez-Sánchez et al.[31]also found that thyme and patchouli oils are the most effective EOs in selected EOs because of the significant reduction of biofilm viability.However,no one EO could completely eradicateStaphylococcus aureusbiofilms at the doses tested(0.1%-8.0%,V/V).

A single essential oil component(EOC)is not enough for inhibiting biofilm growth.Alni et al.[32]reported combinedC.cyminumandA.sativumEOs can be considered as the potential agents against planktonic and biofilm forms ofS.typhimurium.Those data suggested that a single EOC could not restrain biofilm growth effectively since the antimicrobial effects of EOs attribute to the interaction between all of the EOCs rather than the action of an individual component.To effectively control the formation of biofilm,combining EO-based treatments with other bactericides is preferable.

Fig.1.The strategies for controlling biofilm formation.

EOs are also used in medical equipment for the control or elimination of biofilms,dental implants,and other applications[33,34].A study found that clove essential oils and thyme essential oils could inhibit the formation of the bacterial biofilms by preventing planktonic cell attachment on soft contact and by inhibiting the development of the biofilm[35].Nithyanand et al.[34]first reported a new natural resource for EOs extracted fromPogostemonspecies,which showed good anti-biofilm activity for drug-resistant bacterial biofilms.The mechanism of anti-biofilm activity is assumed that the hydrophobic structure of EOs act by separating the lipids from the bacterial cell membrane,which results in the collapse of the bacterial cell structures.On the whole,EOs perturb the membrane and wall of bacterial cells,thus,resulting in structure cleavage and cellular substance leakage[36,37].Furthermore,some EO components affect the fatty acid membrane of the bacterial biofilm,thereby inhibiting biofilm formation[38,39].For example,the components of sesquiterpenes and other phenolic compounds destroy the lipid bilayer by damaging the fatty acid membrane[40].Additionally,phenolic compounds can alter the fluidity,permeability,and composition of a bacterial cell,as well as disturb metabolic pathways(such as depletion of intracellular adenosine triphosphate(ATP)and inhibition of adenosine triphosphatase and gene responses)[41,42].It is suggested that the fatty acid pathway is an attractive target for inhibiting biofilm formation.This indicates that it is more effective to control the biofilm by targeting a certain structure rather than removing the whole formed biofilm.However,EOs have limited application in the food industry due to their volatility and poor solubility,as a result,EOs may be administered internally in a microencapsulated form.

3.2.Enzymes

Enzymes or proteins are biologically active macromolecules that are believed to remove biofilms since proteases or other-degrading enzymes have shown ability in inhibiting biofilm formation[43,44].In fact,in food-processing equipment,such as food surfaces,heat exchangers,and so forth,the function of multiple-specificity hydrolytic enzymes in degrading bacterial biofilm has been demonstrated.Kaur et al.[45]found that the enzyme cocktail fromAspergillus nigerhad a noteworthy potential to eradicate/disperse the biofilms of selected pathogens.The combination of cellulase followed by cetyltrimethyl ammonium bromide(CTAB)immersion showed an obvious effect on removing the mature biofilm[16].During meat processing,it can be selected as a potential reagent for eliminatingSalmonellabiofilm in the maturation step.Moreover,papain disintegrates substances(carbohydrate,protein)ofAcinetobacterandS.aureusforming biofilms[46].Interestingly,the study evaluated the inhibitory properties ofFlavourzyme,a commercial peptidase,againstSalmonella entericaand Shiga toxinproducing(or verotoxin-producing)Escherichia coli.The result found that 4.0 and 5.5 log inhibition of biofilm formation byS.typhimuriumandE.colirespectively when treated with sub-minimum inhibitory concentrations of Flavourzyme for 24 h,which was due to Flavourzyme significantly suppressed the relative expression levels of biofilmforming,quorum sensing,and virulence regulatory genes[26].That was to say,enzymes,as a preventive agent against biofilm formation,possibly played a part in inhibiting bacterial self-defense mechanisms and following disrupting cellular proteins.Bacterial biofilms show great resistance to common disinfectants during food processing.Furthermore,the effect of polysaccharidases and proteolytic enzymes on the bacterial biofilm was studied.Comparison with the results of the EPS composition index and the biofilm removal index revealed that specific proteolytic enzymes,serine proteases were more obvious in removingBacillusbiofilms than the polysaccharidases.On the contrary,for the elimination ofPseudomonasfluorescensbiofilms,the effect of polysaccharidases was more obvious than that of serine proteases[47].Additionally,among the enzyme,the antimicrobial peptide 1018-K6 showed an impressive rapid mode of eradicatingStaphylococcus aureusestablished biofilms within a few minutes and the bactericidal activity against planktonic cells was also observed,which made it a promising candidate for applications in food safety and quality control[48].In short,the use of enzymes can disintegrate the substances of bacterial biofilms and promote the sensitivity of biofilms to hydrodynamic stresses,giving enzymes potential use in the food industries of clean-in-place procedures[25].

Enzymes and their mixtures can target the bacterial biofilm matrix because of the variety of polymers in the matrix[6].The enzymes decrease the adhesion of the EPS in the bacterial biofilm[45].Further,the enzymes disturb the network formed by the EPS[49].Therefore,destroying the physical integrity of the biofilm matrix would be an attractive alternative for both medical and industrial applications.

3.3.Biosurfactants

Biosurfactant is a kind of metabolite with surface activity and amphipathicity that is secreted by microorganisms during metabolism.The biosurfactant has the capability to decrease interfacial tension and allow micellar structure formation[50].As biosurfactants are easily biodegradable and have low toxicity,there is increasing interest in surfactants[51].Studies have found that biosurfactants also reflect antimicrobial ability,and that they further prevent the bacterial cells from attaching during biofilm formation on food-contact surfaces[52,53].It was found that in both static and flow conditions,sophorolipids could inhibit the formation of bacterial biofilms produced by the single or mixed cultures ofBacillus subtilisBBK006 andS.aureusATCC 9144 when they reach 5%(V/V)concentration[54].This result indicated that the sophorolipids may promise adjuvants for other antimicrobials used against bacterial biofilm formation or disruption in biomedical applications.In contrast to rhamnolipid,cetyltrimethyl ammonium bromide and sodium dodecyl sulfate inhibit biofilm formation on stainless steel,in which the biofilm was produced by the seven mixedSalmonellastrains cultivated in bouillon culture medium[16].Hence,research into biosurfactants needs to be strengthened.Kuyukina et al.[54]evaluated the effect of trehalolipid biosurfactant on the adhesion of cells and the formation of biofilms on the polystyrene microplate surface.It indicated that the biosurfactant inhibited the adhesion of bacteria independently of their surface charges.In addition,the effect of diverse anti-adhesive agents depends on the concentration,biosurfactant structure and bacterial species[55].Rhamnolipids and surfactin could reduce the adhesion ofL.monocytogenesandP.fluorescenson polystyrene by up to 79%and 54%respectively;meanwhile,they decreased biofilm formation on stainless-steel surfaces by up to 83%and 73%,respectively[21].The EPS provides mechanical stability for biofilms,regulates their adhesion to surfaces,and forms a cohesive,three-dimensional polymer network that interconnects,transiently immobilizes,and entraps biofilm cells[56].Furthermore,another experiment showed that the system(the outer layer of nanoparticles with mixed lipids of rhamnolipid and phospholipids,the inner core with pectin sulfate)was able to disrupt significantlyHelicobacter pyloribiofilm by eliminating the EPS,and by inhibiting the adherence and colonization of bacteria[57].At present,a biofilm formation inhibitor composed of surfactant is applied in detergents for food or beverage processing plants and medical instruments.

The surfactants can also disrupt the hydrophobic interactions involved in biofilm matrix cross-linking[47,58].The surface hydrophobicity parameter is considered to influence the biofilm formation in the second stage(reversible/irreversible)of microbial adhesion[59,60].Surfactants have been proven to affect the interaction between bacterial cells and the attachment of bacterial cells to surfaces[61].Therefore,effective surfactants are suggested as a potential antimicrobial agent combined with antibiotics for decreasing adhesion and disrupting biofilms.

3.4.Others

Other antimicrobial agents have also been used to study the inhibition of biofilms.Among them,the recent treatment method based on antibacterial photosensitization is considered a new technology for the decontamination of bacterial biofilms during food processing and environment handling[6].The cell-to-cell communication termed“quorum sensing”has been implicated in the formation of biofilm in foodborne pathogens,and many members of theVibrionaceaeare known to regulate activities such as biofilm formation by quorum sensing[3,62].For instance,quorum sensing regulates the secretion of EPS required for biofilm formation inV.cholera[63].Hence,the present study dealt with the effect of signaling molecules on initial adherence and EPS production in biofilms,which are the two major steps required for the initiation of biofilms[3].Anti-quorum-sensing strategies are quite reliable for preventing biofilm formation;they inhibit the swarming motilities that play an important role in biofilm formation[29,64].Cui et al.[65]found that nanoliposomes prepared by encapsulating salvia oil(SO)exhibited antibacterial properties under conditions in milk containers.The reason was the damaging effect of the pore-forming toxin on cell membranes;α-toxin secretedS.aureustriggers the release of SO from nanoliposomes.The microsized ZnO spheres showed the wide-spectrum antibacterial property and antibiofilm activity,especially forS.aureus,B.subtilis,Escherichia coli,Klebsiella pneumonia,andP.aeruginosa[66].It suggests that a new potential ZnO-based antibiofilm formulation can be considered.

The genetic approach can be used to control the expression of genes,which regulate the bacterial adhesion in the early stage and the biofilm formation stage to reach the preventive level.It can also limit the expression of genes involved in bacterial detachment and dispersal during the last step of biofilm formation[67].The genome sequence namelyicaA,D,B,Care associated with the biofilm formation inStaphylococcus aureus[68].Similarly,theCdrAgene promoted biofilm formation in these non-Psl dominantP.aeruginosastrains[69].Similarly,Hu et al.[70]demonstrated thatbcsAandbcsBmutants did not produce cellulose.In contrast to the wild type,thebcsAandbcsBmutants showed the ability to reduce biofilm formation and bacterial cell-cell aggregation in calcofluor binding assays.As the formation and maturation of biofilm are regulated by multiple genes,studies on biofilms are mainly based on gene species and regulation factors.

Adenosine triphosphate(ATP)can stimulate cell lysis and extracellular DNA release,which play a role in cell adhesion during bacterial biofilm formation.Thus,a new strategy is developed to control bacterial biofilm formation.CeO2-decorated porphyrin-based metal-organic frameworks(MOFs)were prepared,and CeO2nanoparticles were released from the MOFs;these inhibited extracellular ATP(eATP),which prevented cell adhesion during bacterial biofilm formation.Additionally,cytotoxic reactive oxygen species released from the MOFs killed the planktonic bacteria[71].The CeO2-decorated MOFs inhibit biofilm formation by depleting bacterial-adhesion-related molecules.The synergy between eATP inhibition and celladhesion disruption effectively prevents biofilm formation and provides a new design for biofilm-inhibiting systems.Furthermore,the flagellar motility and some virulence factors in bacteria likeL.monocytogeneshave been implicated in biofilm formation.The soluble bioactive or metabolic substances naturally produced by these microorganisms,especially the metabolic byproducts produced by probiotics might provide positive effect to the bio-interventions for controlling biofilm.Hossain et al.[9]researched the efficacy of postbiotics collected from isolatedLactobacillus curvatusB.67 andLactobacillus plantarumM.2 againstListeria monocytogenesin planktonic cells and biofilm states.It was found that postbiotics fromLactobacillusspp.could be used as effective bio-interventions for controllingL.monocytogenesbiofilm in the food industry through inhibition on the swimming motility and expression of various target genes associated with pathogenicity and biofilm formationL.monocytogenes.Certainly,it requires further research as known bacterial biofilm formation process.Herein,the inhibitive ability of postbiotics against biofilms formed on food contact surfaces should be verified in food processing facilities to confirm its applications in food safety.

The biofilm is difficult to be eliminated only by inefficient physical treatment methods.Molecular and physiological aspects of biofilm formation related to strainspecific responses.Current and future advances in systems biology and omics technologies are expected to aid significantly in the biofilm formation variability,allowing for its integration in microbiological risk assessment[72].The mechanisms of adhesion and dispersion on dynamic biofilm processes need to be discussed in depth.The above discussions show a comprehensive analysis of new green strategies for inhibiting biofilm formation and scavenging formed biofilms in food industries.It is helpful for effective methods and new strategies to control the dynamic process of biofilm formation in various working processes.

Biofilm formation and foodborne pathogen removal during food processing is a new topic in food safety.Bacterial biofilms related to dairy products,meat,and others in food industries have been studied.However,there is still a major problem in controlling biofilms formation in the food industry.Research has mainly focused on bacterial biofilm morphology and inhibition methods for bacterial biofilm formation.According to previous studies,effective control methods for bacterial biofilm formation during the dynamic processes that have been reviewed include initial irreversible attachment,formation,and maturation,which may help broaden microbial food safety research.The biosensor which can alter a biological signal into the measured signal is expected to exhibit a rapid and broad-spectrum activity to develop anti-biofilm resistance.The anti-biofilm peptide may be a good candidate in aqueous solutions such as the implementation of“green”packaging technologies and bio-sanitizing formulations.With the arrival of the post-genome age,more chances for the study of related microbial levels can be obtained through genomics,proteomics,and metabolomics.At the same time,the kinetic process of bacterial biofilm formation is necessary to study the mechanisms of biofilm removal deeply.

Author Contributions

Tingwei Zhu:Writing-original draft,writing-review&editing;Chenxian Yang:Supervision;Xuerui Bao:Supervision;Fusheng Chen:Validation;Xingfeng Guo:Validation.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was funded by the Science and Technology Project of Henan Province(212102110320),and the Science Foundation of Henan University of Technology(Grant Nos.2020BS013;2019BS029).We would like to thank International Science Editing(http://www.internationalscienceediting.com)for editing this manuscript.