Natural antimicrobial-coated supports as filter aids for the microbiological stabilisation of drinks

The strategy of applying surface modifications to inert supports to improve their physicochemical, biological and functional properties can be adopted to develop novel filtering materials by modifying surfaces of widely used materials with biocompatible antimicrobial molecules. This approach would enable overcoming the limitations of conventional filtration methodologies in water treatment and the microbial stabilisation of drinks. The co-valent immobilisation of naturally-occurring antimicrobial compounds on filter surfaces allows the antimicrobial effectiveness of the anchored molecule to be preserved by conferring the support material antimicrobial and antifouling properties. This review focuses on describing the naturally-occurring antimicrobials used in the food industry to either prevent growth or inhibit microorganisms, and defines the most established immobilisation methodologies by reviewing the different materials that can be used as grafting supports, and providing details of some examples of naturally-occurring antimicrobial compounds immobilised on filter aids applied to microbi-ologically stabilise beverages. Besides overviewing the most recent research, this work discusses the advantages, limitations and challenges of developing natural antimicrobial-coated filter aids, and it points out the innovation potential beyond current research on different supports and applications. Before such aids are applied to a real environment, more knowledge on safety and manufacturing is needed on the industrial scale. Therefore, the information in this review may be valuable to extend new emerging preservation technologies to control the food and drink microbial contamination that results in minimally processed products.


Introduction
The covalent immobilisation of antimicrobial molecules onto substrate surfaces is an approach that allows materials with additional functionalities to be developed, while maintaining bulk properties, which can be used in many technical fields (Treccani et al., 2013).In the food industry, antimicrobial-coated materials can be applied to different food processing stages because immobilised antimicrobials exert their biocidal properties after grafting and do not significant affect food properties because of their immobilisation (Ruiz-Rico et al., 2017).Indeed the use of natural antimicrobial-coated supports as filtering materials can potentially be applied as a food preservation technique (Cappannella et al., 2016;Peña-Gómez, Ruiz-Rico, Fernández-Segovia, & Barat, 2019;Zhang et al., 2021).
Filtration as a separation technique is applied to remove suspended particles from a liquid by passing them through a filter medium.Different filtration processes, including depth filtration and membrane filtration, have been used as beverage stabilisation techniques.Despite their removal properties, they involve clogging problems, require regeneration techniques, and their ability to eliminate certain pathogens is limited (Gibert et al., 2013).Therefore, enhancing the effectiveness of the common filters used in water and food treatments by developing antimicrobial-coated supports can address the challenges that current technologies face, and confer the capability to stabilise and eliminate microorganisms from drinks without affecting food properties.
This review focuses on describing naturally-occurring antimicrobials covalently immobilised on materials' surfaces to develop antimicrobialcoated supports to be used as filter aids to microbiologically stabilise beverages.A literature search in the databases Web of Science® and Scopus® was conducted with the following keywords: natural antimicrobial, immobilisation, coating, functionalisation, filtration, water, drink.Combinations of these keywords identified relevant studies in the field.Over a hundred articles were collected, but selection criteria were applied to focus this review on research on the immobilisation of natural biocidal molecules and the application of natural antimicrobial-coated supports in the drink industry.

The naturally-occurring antimicrobial compounds to be immobilised
Natural antimicrobials are defined as biomolecules with inhibitory activity that are obtained from various sources in nature, including those of plant, animal and microbial origins (Lucera, Costa, Conte, & Del Nobile, 2012).Increasing attention has been paid to the use of natural antimicrobials for food biopreservation purposes thanks to their ample biocidal activity (Pisoschi et al., 2018).Details of their origin and mode of action are summarised in Fig. 1.Their direct application is limited by interaction with food components, instability and impact on food properties (Abdelhamid & El-Dougdoug, 2020).This is why their covalent immobilisation on supports has been proposed to overcome current limitations with their use in free forms, but it allows the resulting antimicrobial-coated materials to be applied in new technical fields.To better understand the potential immobilisation techniques that can be applied to the different natural antimicrobials, the next subsections describe their main properties.

Essential oils and other phenolic compounds
Essential oils and other aqueous extracts are antimicrobials of plant origin generated by plants as a protection strategy (Burt, 2004;Daglia, 2012).Although their mechanism of action is not altogether clear, it is related to their chemical structure, particularly to the presence of hydrophilic functional groups like hydroxyl groups and substituents on the aromatic ring, the number of double bonds and/or lipophilicity (Lucera et al., 2012).The interaction of these molecules with lipids of microbial membranes enhances their permeability, which causes cytoplasmic content to leak and leads to cell death.They can also interfere with membrane proteins by inducing their alteration, which affects electron transport, nutrient uptake, synthesis of proteins and nucleic acids, and enzyme activity (Pisoschi et al., 2018).
Their direct application to food can affect food properties, mainly sensory properties, and diminish efficacy due to food matrix factors, such as fat content, proteins, water activity, pH and enzymes (Lucera et al., 2012).They can also present environmental instability and low water solubility, which make their dispersion in food difficult (Burt, 2004;Vico et al., 2016).

Fatty acids
Medium-chain saturated and long-chain unsaturated fatty acids are powerful biocidal agents.Lipids inactivate microorganisms primarily by disrupting the bacterial cell membrane or inhibiting other intracellular targets like hindering DNA replication (Pisoschi et al., 2018).
The disadvantages of using free fatty acids are instability and interaction with food components, which can reduce their availability, along with their impact on food organoleptic and functional properties (Desbois & Smith, 2010).

Polysaccharides, proteins, peptides and enzymes
Chitosan is a biodegradable and non-toxic biopolymer with antimicrobial properties.Its mechanism of action is related to its polycationic structure, which acts as a water-binding agent, an enzyme inhibitor and a bioabsorbant of nutrients, and improves membrane permeability and hampers mRNA and protein synthesis (Pisoschi et al., 2018).Application to food is limited given its scarce solubility at neutral and alkaline pH values.
Lactoferrin is an iron-binding glycoprotein present in milk or other secretions (saliva or tears) with good antimicrobial activity.Lactoferrin binds two atoms of iron, which has been related to the antibacterial effect due to iron sequestration.Other mechanisms might also be involved, such as the blocking microbial metabolism of carbohydrates or destabilising the bacterial cell wall (Harouna et al., 2015).
Other antimicrobial proteins, like avidin and ovotransferrin, and peptides like pleurocidin, defensins, protamine, magainin, and casocidin, present a wide range of antibacterial activities (Quinto et al., 2019).The mechanism of action of these peptides is related mainly to their amphipathicity, which allows the electrostatic interaction with the microbial membrane to result in membrane permeabilisation and bacterial osmoregulation alteration (Li et al., 2021).Antimicrobial peptides have some interesting features such as biocompatibility, low toxicity, broad antimicrobial spectrum and selectivity towards specific multi-drug resistant species.However, they also present some limitations mostly limited efficacy in vivo due to reduced bioavailability and high production costs (Hazam, Goyal, & Ramakrishnan, 2019).
Lysozyme is a lytic enzyme found in foods (milk and eggs) or human Fig. 1.Classification and mode of action of naturally-occurring antimicrobial compounds according to their source in nature.
M. Ruiz-Rico and J.M. Barat secretions.It presents antimicrobial properties mainly against Grampositive bacteria because their cell wall consists of peptidoglycans that are hydrolysable by this enzyme, which results in cell lysis (Pisoschi et al., 2018).Enzymes are sensitive to production and storage processes, and can interact undesirably with food components, which can decrease their antimicrobial efficacy (Lopes, Barreto Pinilla, & Brandelli, 2019).

Bacteriocins
Bacteriocins, such as nisin, natamycin or pediocin, are small bacterial peptides with excellent antimicrobial activity.Their antimicrobial activity is due to an alteration to membranes by electrostatic interactions with negatively charged membrane phospholipids, which results in both the loss of intracellular components and in proton motive force disruption (Pisoschi et al., 2018).
Their effectiveness is conditioned by external factors like pH, temperature, composition, structure and microbiota of food.Interaction with food components or microbial load present in food can result in precipitation, degradation or inactivation of bacteriocins (Gálvez, Abriouel, López, & Omar, 2007).

Bacteriophages
Lytic bacteriophages have been proposed as natural antimicrobials to control foodborne pathogens in food.These viruses only infect bacteria and can inhibit specific pathogenic bacteria.However, the ability of microorganisms to develop immunity to phages can create resistance by undermining their application (Quinto et al., 2019).

Natural antimicrobial-coated filtering materials to stabilise drinks
Surface coating strategies have been developed to immobilise bioactive compounds on materials that can be potentially applied to diverse industrial areas (Treccani et al., 2013).The aim of applying this approach is to modify the properties of bulk materials, and can be applied to common filters to confer them new properties.There are many examples in the literature of such that describe the immobilisation of silver or titanium dioxide nanoparticles, often used as water disinfectants (Hossain, Perales-Perez, Hwang, & Román, 2014), on filters applied to water treatment (Lalley et al., 2014).However, they are not natural antimicrobials and are not, therefore, the aim of this review.In contrast, some examples exist of natural antimicrobial-coated filters applied to microbiologically stabilise water or other beverages.So apart from describing and discussing these natural antimicrobial-coated filters, some examples of covalent immobilisation of natural antimicrobials on diverse supports with preserved biocidal properties are included in this review to provide a more complete picture of the enormous possibilities of covalent immobilisation of natural biomolecules that may be applied to develop filtering materials.This work also briefly discusses the main properties that support materials should have and the principal immobilisation techniques.

Properties of support materials used for immobilisation
Biomolecule immobilisation depends on the features and properties of the material used as a support.This support should meet some requirements to make it suitable for immobilisation: (i) relatively inexpensive and environmentally harmless; (ii) good thermal, chemical and mechanical stability; (iii) microbial attack resistance; (iv) inert to immobilised biomolecules; (v) a large surface area and a proper particle size; (vi) presence of enough functional groups for biomolecule grafting (Zucca & Sanjust, 2014).
Supports can be classified as organic and inorganic substrates according to their nature.Of organic materials, cellulose is considered to be non-toxic, cost-effective and biocompatible material (Zdarta, Meyer, Jesionowski, & Pinelo, 2018).Cellulose is a linear polymer of glucose units in which hydroxyl groups are responsible for its chemical activity.However, backbone hydroxyl groups are non-reactive for specific reactions and most cellulose supports need to be activated to make surface functional groups suitable for a subsequent bioconjugation reaction.Chitosan is a linear polysaccharide with several functional groups.The presence of many hydroxyl and amine groups in its structure facilitates the effective grafting of biomolecules without involving any modifying or cross-linking agents (Zdarta et al., 2018).Besides natural organic materials, synthetic polymers like polystyrene, polyethylene, polyamide or ethylene vinyl alcohol, among others, are also used as substrates for biomolecule immobilisation (Goddard & Hotchkiss, 2007).
Ceramic materials are suitable inorganic substrates for biomolecule immobilisation thanks to their excellent mechanical and chemical stability, large surface area, porous structure, controllable shape and size, and the functional moieties (hydroxyl, carbonyl and carboxyl groups) present on their surface (Zdarta et al., 2018).Silica is the most widely used inorganic material for biomolecule immobilisation given its surface hydrophilic character and the presence of many hydroxyl groups that come in the form of silanol groups (Zucca & Sanjust, 2014).Other mineral substrates can also be used for biomolecule immobilisation, including glass, sand, halloysite, kaolin, clays or bentonite (Zdarta et al., 2018).
Metallic materials are inorganic supports with excellent structural, physico-chemical, thermal and mechanical properties, biocompatibility, good durability and high conductivity (Su et al., 2018).Titanium, zinc oxide, alumina, magnetic iron oxide or stainless steel can be used for biomolecule immobilisation (Zdarta et al., 2018).Some of these inert metal surfaces need to be pre-activated to modify their surface prior to immobilisation.

Techniques used for immobilisation
Immobilisation consists of entrapping or anchoring a biomolecule to a support, preserving the bioactive properties of grafted molecules, conferring the support material new properties, and preventing them from continuous circulation in the environment.Immobilisation can be divided into reversible and irreversible techniques according to the interaction between the biomolecule and support.Reversible immobilisation examples include physical entrapment, adsorption or ionic binding, whereas covalent immobilisation is an irreversible methodology.
Covalent immobilisation offers remarkable advantages over reversible immobilisation techniques, including greater stability, longer shelf life, biomolecule metabolism prevention and biomolecule migration inhibition, all because the covalent bond between the functional groups present in the biomolecule and on the support surface does not allow the biomolecule to be released.This review focuses on the covalent immobilisation of natural antimicrobials and, therefore, the fundamentals of this technique are herein reviewed.
Covalent immobilisation is a multi-step process that generally requires the activation of the support's surface, followed by surface functionalisation to add functional reactive groups, and then the activation of reactive moieties and reaction with the biomolecule.
The support has to have functional groups on its surface because otherwise it may be necessary to modify its surface for immobilisation.Surface functionalisation involves introducing a functionalising agent into a surface to tailor the surface properties for a specific purpose.Different techniques, including wet chemical, silanisation and dry oxidation, are used to introduce reactive groups into material surfaces, which serve as an anchorage for immobilisation (Goddard & Hotchkiss, 2007).
Wet chemical modification consists of treating a material with liquid agents to create reactive functional groups on its surface (Goddard & Hotchkiss, 2007).This is an easy technique with excellent penetration capacity.However, it is an irregular functionalisation process, and is not efficient and fast enough due to low reaction kinetics and multiple-step approaches, and it generates hazardous chemical waste (Nayak, Rahaman, & Giri, 2019).
Silanisation involves covering the support's surface with alkoxysilane moieties, which form a coating of covalently bonded silanes on the surface with an organic reactive functional group to enable them to immobilise biomolecules.Selecting the most appropriate organosilane must be done according to the chemistry of the biomolecule that needs to be immobilised.Organosilanes terminated with amine, thiol, isocyanate or carboxyl functional groups are often used in silanisation (Goddard & Hotchkiss, 2007).
Dry oxidation using ozone or reactive plasma provides a highly reactive chemical environment that can change the surface properties of inert materials.Ozone oxidation is an industrially cost-effective technique for generating a variety of oxygen-bearing polar groups (Nayak et al., 2019).Plasma can be used as a precursor to other surface modification techniques like silanisation or UV irradiation.Surfaces exposed to UV light can generate reactive sites for functional groups upon exposure to gas, or can be utilised to initiate UV-induced graft polymerisation (Goddard & Hotchkiss, 2007).
Activation is the final step prior to biomolecule immobilisation when the support's surface is functionalised.This step consists of generating both reactive groups on supports and the biomolecule compatible for the coupling reaction (Zucca & Sanjust, 2014).To convert one functional group into another, or to assist the bioconjugation process, different activation and cross-linking agents (i.e.glutaraldehyde, carbodiimides, etc.) can be used to gain the common functional groups employed in bioconjugation chemistry, such as thiols, aldehydes, carboxylic acids, hydroxyls and primary amines (Goddard & Hotchkiss, 2007).

Covalent immobilisation of naturally-occurring antimicrobial compounds
The covalent immobilisation of natural antimicrobials should result in antimicrobial-coated supports with a contact-active mode of action, and not in the release of a biocidal agent.The literature describes the covalent immobilisation of natural antimicrobials on different supports which, despite most not being applied as filtering materials, are included in this review as examples of possible strategies to develop new antimicrobial-coated filters for beverage microbial stabilisation.
The most important examples of the covalent immobilisation of natural antimicrobials reported in recent years are found in Table 1.It is highlighted that the mechanism of action of the developed antimicrobial supports is due to the immobilised biomolecule's inherent inhibitory properties after microbial cells have come into contact with the antimicrobial, which affects bacterial viability and metabolic activity (Lee & Je, 2013;Muriel-Galet, Talbert, Hernandez-Munoz, Gavara, & Goddard, 2013;Nogueira et al., 2017;Ribes et al., 2017;Yuan et al., 2013).Examples of the covalent immobilisation of natural antimicrobials and their biocidal mode of action are shown in Figs. 2 and 3. Nevertheless, the covalent grafting of these biomolecules on inert supports has distinct advantages over their free-form counterparts.
The irreversible immobilisation of natural antimicrobials can result in systems with enhanced antimicrobial activity.The use of phenolic compounds, such as vanillin or gallic acid immobilised on inorganic supports, has shown that the inhibitory effect of immobilised biomolecules is preserved, and even enhanced (Ruiz-Rico et al., 2017;Vico et al., 2016;Polo et al., 2018).Improved antimicrobial activity is probably due to the presence of high local antimicrobial concentrations on particles' surfaces and, therefore, in the cell's surroundings, which can easily disrupt cell membrane and induce cell death (Fig. 2C) (Ruiz-Rico, Moreno, & Barat, 2020; Vico et al., 2016).
Another advantage of the covalent immobilisation of natural antimicrobials is grafted biomolecules' increased stability.Liburdi, Benucci, Palumbo, and Esti (2016) reported the immobilisation of lysozyme on chitosan beads and showed a reduction in the lysozyme's cell lysis velocity and antimicrobial activity after immobilisation, probably due to diffusion limitation, steric hindrance and coupling via amino acid residues essential for catalytic activity.However, antimicrobial supports presented greater stability than the free lysozyme after being incorporated into wine because enzymatic activity was less affected by wine components.Tavakolian et al. (2018) developed lysozyme and nisin-coated nanocrystalline cellulose supports in which immobilisation reduced the possible spatial orientations of these proteins by restricting their interaction with the microbial membrane and, therefore, reducing inhibitory properties.In contrast, immobilisation allowed the antimicrobials' activity to continue longer.Besides longer stability, covalent grafting allowed resistant antimicrobial-coated supports for several cleaning conditions to be obtained (Duday et al., 2013;Nogueira et al., 2017).
One crucial drawback of some natural antimicrobials like essential oil components (EOCs) is their strong odour and flavour, which limit their use in the food industry.However, their covalent immobilisation on a support can mask the sensory impact of antimicrobials.Ruiz-Rico et al. ( 2017) and Ribes et al. (2017) evaluated the effect of incorporating EOCs-coated silica particles into different food matrices by sensory analyses.These authors confirmed that sensory perception significantly decreases when antimicrobials are covalently immobilised because covalent grafting avoids the release and volatilisation of biomolecules.
The biocompatibility and safety of natural antimicrobials can also be modified after their covalent immobilisation.Chen, Shi, Neoh, and Kang (2009) reported a lower cytotoxicity of carvacrol and eugenol after grafting them onto chitosan nanoparticles using fibroblast cells.In contrast Verdú et al. (2020) reported potential alterations of the toxicological parameters of gallic acid on the in vivo Caenorhabditis elegans system.Immobilised antimicrobial increased nematode mortality, affected locomotion behaviour and disturbed its response to thermal stress compared to the equivalent free gallic acid concentration.In this case, immobilisation was a disadvantage because it blocked gallic acid internalisation, which has a protective effect on nematode viability because of its antioxidant properties.Some of the antimicrobial supports described in this section were designed to be applied as food additives (Lee & Je, 2013;Ribes et al., 2017;Ruiz-Rico et al., 2017), food processing aids (Liburdi et al., 2016), food packaging (Calce et al., 2014;Muriel-Galet et al., 2013) or food contact surfaces (Duday et al., 2013).Nevertheless, the immobilisation approaches described in Table 1 offer remarkable advantages and can be an inspiration to develop new antimicrobial-coated filters for the microbial stabilisation of liquid matrices.

Application of antimicrobial-coated supports as filter aids
The antimicrobial-coated filter aids described in this section (Table 2) are diverse, but share one common feature: the removal of microbial cells is achieved by combining cell retention on the support surface (partial or complete according to the particle or pore size of filters) and the immobilised molecule's biocidal activity, which confers filters: (i) antimicrobial activity based on contact killing and/or; (ii) antifouling activity based on adhesion resistance.
A first approach can be found in the literature: coated filters with antimicrobial activity.The simplest reported design is the adsorption a natural cationic protein of Moringa oleifera seeds onto sand as an antimicrobial flocculant for water clarification (Jerri et al., 2012;Samineni et al., 2019).Antimicrobial sand was able to capture bacteria and virus (7-8 log reduction values (LRVs)) by an electrostatic interaction between the cationic protein and the negatively charged bacteria or virus capsid proteins.These filters showed an extraordinary removal capacity, which can be applied to treat irrigation and drinking water.However, the antimicrobial is not covalently immobilised on the filter and will, therefore, be released to the treated matrix, which can limit its long-term use, safety and applicability.
Apart from adsorption, different filters with antimicrobial activity (2) amide bonding between the amino groups of 3aminopropyltriethoxysilane grafted on the surface of silica nanoparticles and the carboxyl group of gallic acid.
Both antimicrobial-coated particles showed higher antimicrobial activity than free gallic acid against Paenibacillus larvae.

Gallic acid MCM-41 silica microparticles
Amide bonding between the amino group of 3aminopropyltriethoxysilane grafted on the surface of the silica particles and the carboxyl group of gallic acid.
Antimicrobial-coated particles showed enhanced inhibitory effect against L. innocua.The effect of free and immobilised GA on the in vivo system Caenorhabditis elegans revealing alterations of the toxicological parameters of the biomolecule.

Verdú et al. (2020)
Linoleic acid, oleic acid and palmitic acid Pectin Anhydrides of fatty acids were synthesised and then immobilised by acylation of the alcoholic functions of pectin with fatty acid anhydrides by grinding in the presence of the K 2 CO 3 catalyst and ethanol with microwave irradiation.
Fatty acid-coated pectin partially inhibited S. aureus and E. coli.Coatings of pectin derivatives on polyethylene films were applied as an active packaging displaying oxygen scavenging function.

Lysozyme
Chitosan beads Lysozyme immobilisation using glutaraldehyde as crosslinker between the enzyme and the chitosan surface.
Kinetic characterization of lytic reaction and antimicrobial activity of free and immobilised lysozyme using model wine and white wine inoculated with O. oeni showed the reduction of cell lysis velocity and antimicrobial activity after immobilisation.However, the enzymatic activity was less affected by wine components when lysozyme was immobilised.due to natural antimicrobials covalently immobilised on their surfaces have recently been reported (Cappannella et al., 2016;Kroll et al., 2012;Ribes et al., 2020;Wang et al., 2017;Zhang et al., 2021).The most important parameter to be evaluated with these antimicrobial-coated filters is their removal capability and, consequently, their mode of action.Microbial contamination elimination is based on: (i) the retention  After establishing filters' removal capability, relevant operating conditions should be evaluated to validate their applicability.The main limitation with this approach is filters' limited lifetime and coating stability during working operations.Some of the discussed studies have evaluated stability parameters under different conditions and reached ambiguous conclusions.The stability assays of the lysozymefunctionalised membranes reported by Kroll et al. (2012) showed that enzyme activity is affected by flow conditions and results in: (i) diminished removal at high flow rates because the contact time between the immobilised lysozyme and the bacterial cells in solution substantially shortens; (ii) immobilised molecule leaching because of the enhanced shear stress near the microtube surface.Peña-Gómez et al. (2019a, 2019b, 2020) determined the potential leaching of immobilised EOCs from silica supports after single filtration or pre-conditioning.According to the immobilised biomolecule, they found diverse wash-out effects, most of which were probably related to the immobilisation procedure rather to than the operating conditions.Therefore, a key parameter to be addressed in future works is to optimise the immobilisation procedure to ensure antimicrobial activity maintenance and zero wash-out in long-term uses.Zhang et al. (2021) assessed the stability of nisin-coated microfiltration membranes with different cleaning protocols (0.02 M HCl, deionised water, 0.001 M NaOH and 0.02% dodecyl sodium sulphate), which resulted in acceptable membrane stability.However, long-term use and successive cleaning cycles should be evaluated after establishing suitable cleaning/regeneration procedures.
The impact of the matrix composition on filters' removal capacity is another important parameter that determines shelf life, and this highlights the need to test the developed materials under conditions relevant to the final application (site-, condition-and matrix-specific).Cappannella et al. ( 2016) developed lysozyme-functionalised chitosan spherical supports to produce an effective fluidised bed system for wine microbial stabilisation.Covalent immobilisation significantly reduced lysozyme antimicrobial activity, probably because of the blockage of some active sites during coupling (Liburdi et al., 2016), but enzyme stability increased, which prolonged the useful life for industrial uses.The fluidised bed system was able to lyse the bacteria inoculated in wine more efficiently than the free lysozyme.This represents the notion that covalent immobilisation prevents enzyme inactivation in the presence of wine components.Other studied coated filtering materials with different liquid matrices (water, beer, apple juice) are silica microparticles covalently coated with EOCs for depth filtration (Peña-Gómez et al., 2019a, 2019b, 2020;Ribes et al., 2020).These coated silica filters presented a removal capability of 4-5 LRVs against different microorganisms naturally present or inoculated in diverse matrices, and their removal capability was maintained after filtering multiple samples and pre-conditioning (washing with water) (Peña-Gómez et al., 2019b, 2020).The removal capability of the developed supports on real drink matrices is equivalent to other preservation techniques, which confirms their potential use as an alternative methodology to microbiologically stabilise beverages.
However, the filtration impact on drink properties should also be evaluated to validate its applicability as a preservation technique.No reported study has assessed this effect, except that by Peña-Gómez et al. (2019a), who examined the filtration impact through EOC-functionalised filters on apple juice properties and shelf life.The use of these filters for juice cold pasteurisation allowed drink properties to remain or only had a slight effect if partial antimicrobial leaching occurred.
In addition to the considered parameters, one major current limitation of the developed materials is that no studies have worked under optimised process conditions to simulate real environments, which limits results being extrapolated to the drink industry.Besides, natural antimicrobial-coated filters should be tested with different liquid matrices and against several microorganisms (spoilage and pathogen microorganisms, vegetative cells, spores) to validate their efficacy.
As a second approach, natural antimicrobial-coated filter aids with both antifouling activity and antimicrobial activity can be found.Small

Lysozyme
The membranes were modified by wet chemistry and ethylenediamine to incorporate amino moieties on the support's surface and lysozyme was covalently immobilised using glutaraldehyde as coupling agent.
Water Lysozyme-coated membranes exhibited antibacterial performance against S. aureus by the inhibitory activity of immobilised lysozyme resulting in membranes with antifouling properties.

Lysozyme
Lysozyme was covalently grafted on the chitosan beads by using a coupling agent containing aldehyde groups that produced the cross linking between the aldehyde groups of the coupling agent with the amino groups of the chitosan surface and the amino groups of the lysozyme by Schiff base reaction.

White and red wine
The use of the coated supports in a fluidised bed system for wine microbial stabilisation allowed the continuous lysis of O. oeni inoculated in white and red wines in a more efficiently way than the free lysozyme and without leaching on the immobilised enzyme.Immobilisation increased the enzyme stability in batch, increasing the useful life for industrial use., Mohanraj, Kandiyote, Kasher, and Arnusch (2018).As previously explained, the aim of the immobilisation of natural antimicrobials on such filters is to incorporate an antifouling agent to design self-cleaning filters.Therefore, the removal capability parameter is not often evaluated in these filters, while permeate flux and salt rejection are essential factors for reverse osmosis membranes.The antimicrobial-coating moderately affected these parameters, but enhanced their long-term use by avoiding biofilm formation (Mao et al., 2018;Saeki et al., 2013).These antifouling membranes could address the persistent challenge of biofilm blocking in water purification technology and may be applicable to other filtration systems to increase the efficiency and useful life of filters.However, performance under the operating conditions of a real environment, the possible interaction of matrix components in adhesion-resistance capacity and potential leaching should be evaluated before real applications.The antimicrobial-coated filters described in this section correspond to a laboratory development stage, and provide very little insight into practical aspects, such as scaling-up production, long-term stability or performance under real operating conditions.Only after evaluating these parameters can realistic production cost estimations be made.Therefore, before being applied to a real scenario, it is necessary to perform additional semi-pilot and pilot scale studies closely related to final industry applications to eliminate contradictory results and to overcome current limitations.Last but not least, the safety of the developed materials should be particularly considered.The use of covalent immobilised antimicrobial-coated supports limits these materials' toxicity risk, but the impact of potential bond destabilisation leaching should be thoroughly evaluated.The immobilisation procedure  should be investigated in-depth for maximum effectiveness and zero leaching purposes.Besides, regulations that deal with the handling and discharge of these new materials should be established to estimate possible risks and to minimise environmental impacts.

Concluding remarks and future perspectives
Despite the effectiveness of today's preservation methodologies, huge efforts are being made to find suitable non-thermal methodologies capable of preserving food properties and delivering microbiological safe products.Of them all, the use of natural antimicrobials immobilised on supports as processing aids (filters) has the potential to stabilise and eliminate microorganisms from beverages.
By tailoring the filter surface with natural antimicrobials, a wide portfolio of supports can be developed to be applied to diverse liquid matrices.The covalent immobilisation of antimicrobials on filters allows bioactive properties to be maintained without releasing the compound to the environment and conferring filters antimicrobial and/or antifouling properties.Therefore, antimicrobial-coated filters can increase not only the existing technological efficiency of current filtration methodologies, but also the useful life of filters, which cut drink treatment costs.Although surface modification techniques are expensive, their advantages may surpass production costs if the design of efficient natural antimicrobial-coated filters with long-term stability is possible.
Although major knowledge gaps for the technical development and safety of natural antimicrobial-coated filters remain, their remarkable properties indicate that they can play a key role in drinks preservation in the future.They can be used as supports in different filtration techniques (standard filtration, microfiltration, nanofiltration, ultrafiltration, reverse osmosis) by modifying material properties like thickness, particle/pore size, geometry or dimensions, which extend potential applications for different matrices and in distinct areas of interest (food, biotechnology, chemistry, environmental remediation, etc.).The combination of more than one immobilised antimicrobial on the same surface also extends their application range, and this technique can be combined with other existing mild-thermal and non-thermal preservation methods to open up new pasteurisation and sterilisation possibilities.Finally, immobilisation techniques followed to develop coated filters can be applied to create antimicrobial-coated surfaces with inhibitory properties by contact or antifouling activity, which can be used in many fields (food, biomedical, textile, etc.).
base reaction via imine bonding between the amino groups of lysozyme or nisin and the aldehyde Lower antimicrobial activity of immobilised compounds than free Tavakolian et al. (2018) (continued on next page) M. Ruiz-Rico and J.M. Barat

Fig. 3 .
Fig. 3. (A) Schematic procedure of lysozyme and chitosan immobilisation on stainless steel surfaces according to Yuan et al. (2013).(B) Mode of inactivation of microbial contamination after coming into contact with immobilised antimicrobials.

Fig. 4 .
Fig. 4. (A)Schematic lysozyme immobilisation procedure applied to zirconia membranes according toKroll et al. (2012).(B) Lysozyme-coated membranes were used in microfiltration to enhance microbial removal by inactivation after coming into contact with the immobilised antimicrobial.

Fig. 5 .
Fig. 5. (A) Schematic carvacrol immobilisation procedure applied to amorphous silica microparticles according to Peña-Gómez et al. (2019b).(B) Essential oil components-coated silica microparticles (5-50 μm) were used as filters in bed filtration to enable superior microbial removal by a combination of physical adsorption on supports and inactivation after coming into contact with immobilised antimicrobials.(C) SEM images of Escherichia coli after filtering through the bed of non-coated and antimicrobial-coated particles.

Table 1
Examples of naturally-occurring antimicrobial compounds covalently immobilised on the surfaces of different support materials.

Table 2
Summary of recent examples of applications of natural antimicrobial-coated supports as filter aids in water and drink matrices.