Héctor Gómez-Llorente,Isabel Fernández-Segovia,Édgar Pérez-Esteve,Susana Ribes,Alejandro Rivas,María Ruiz-Rico,José M. Barat
Immobilization of Natural Antimicrobial Compounds on Food-Grade Supports as a New Strategy to Preserve Fruit-Derived Foods
naturally occurring antimicrobials,covalent immobilization,food preservatives,processing aids,juice,jam,wine,soft drink
The use of natural antimicrobials in the food industry is being proposed as an eco-friendly postharvest technology to preserve fruit-derived foods. In this context, this systematic review aims to describe and discuss the application of naturally occurring antimicrobial compounds in the processing of fruit-derived foods by the PRISMA methodology. In a first step, the use of free natural antimicrobials was investigated as an approach to identify the main families of bioactive compounds employed as food preservatives and the current limitations of this dosage form. Then, the use of immobilized antimicrobials, in an innovative dosage form, was studied by distinguishing two main applications: addition to the food matrix as preservatives or use during processing as technological aids. Having identified the different examples of the immobilization of natural antimicrobial compounds on food-grade supports, the mechanisms of immobilization were studied in detail to provide synthesis and characterization guidelines for future developments. Finally, the contribution of this new technology to decarbonization and energy efficiency of the fruit-derived processing sector and circular economy is discussed in this review.
Immobilization of Natural Antimicrobial Compounds on Food-Grade Supports as a New Strategy to Preserve Fruit-Derived Foods

The use of natural antimicrobials in the food industry is being proposed as an eco-friendly postharvest technology to preserve fruit-derived foods. In this context, this systematic review aims to describe and discuss the application of naturally occurring antimicrobial compounds in the processing of fruit-derived foods by the PRISMA methodology. In a first step, the use of free natural antimicrobials was investigated as an approach to identify the main families of bioactive compounds employed as food preservatives and the current limitations of this dosage form. Then, the use of immobilized antimicrobials, in an innovative dosage form, was studied by distinguishing two main applications: addition to the food matrix as preservatives or use during processing as technological aids. Having identified the different examples of the immobilization of natural antimicrobial compounds on food-grade supports, the mechanisms of immobilization were studied in detail to provide synthesis and characterization guidelines for future developments. Finally, the contribution of this new technology to decarbonization and energy efficiency of the fruit-derived processing sector and circular economy is discussed in this review.

The consumption of natural healthy products, such as fruit and their derivatives (i.e., juices, soft drinks, jams, sauces, or wines), has increased in recent years owing to their convenience and acknowledged nutritional and functional properties. These properties are the result of their high content of vitamins (A, C, and B group), antioxidants, polyphenols, and fiber, and their low sodium and fat contents [1]. As fruit-derived foods are perishable, their processing plays a crucial role in guaranteeing their safety and extending their shelf life. Fruit preservation techniques are based mainly on the use of heat treatments or synthetic preservatives. Heat treatments (i.e., pasteurization and sterilization) enable a product’s microbial load to be reduced or eliminated, and allow the enzymes present in food to be deactivated and, thus, contribute to greater stabilization. Thermal pasteurization is considered the most appropriate methodology for some fruit-derived foods, such as juices, to ensure a 5-log reduction in the microorganisms that can cause spoilage (i.e., Alicyclobacillus acidoterrestris) and pose public health problems (i.e., Escherichia coli O157:H7 or Salmonella enterica) according to the U.S. FDA (Food and Drug Administration, 2001). However, heat treatments cause loss of water-soluble and oxygen-labile nutrients, such as vitamins [2], and undesirable organoleptic changes such as reduced fresh-like flavor [3]. Another relevant factor to consider is the preservation methodology’s carbon footprint. Heat treatments use gas or fuel as a source of energy and release greenhouse gas (GHG) emissions. Consequently, they contribute to global warming. As a piece of data, it can be stated that GHG emissions from juice pasteurization operations amount to 5.5 g CO2/L of juice in a 96% heat recovery thermal system [4]. Considering that in most cases juice is pasteurized at least twice (once immediately after extraction and again prior to bottling), the result is 11 g CO2/L of juice. With these data, the carbon footprint that results from the pasteurization of liquid foods in Europe can be estimated at 1 × 105 tons CO2/year. Moreover, the addition of synthetic preservatives, such as sorbates, benzoates, or sulfur dioxide, is very much questioned because their use can lead to dangerous health problems or antimicrobial resistance [5], and such preservatives can be rejected by consumers for not being natural [6]. In order to reduce the impact of thermal processing on the carbon footprint, and also on the sensory, nutritional, and functional properties of fruit-derived foods, different cold pasteurization methods have been developed as an alternative to conventional preservation methodologies [7]. These methods can be grouped into two groups: (i) physical methods, such as UV irradiation, high-pressure processing, high-intensity pulsed electric field, ultrasounds, filtration, etc., and (ii) chemical methods. Non-thermal physical treatments have been developed to preserve the sensory and nutritional properties of foods. However, they are ineffective against bacterial spores, are expensive, require complex equipment and management procedures, and can even have a bigger carbon footprint than conventional heat treatment [4]. In the application of unconventional chemical methodologies, it is important to highlight the use of naturally occurring antimicrobial compounds to ensure food safety and quality due to their broad-spectrum antimicrobial activities and biocompatibility [8]. This review focuses on employing naturally occurring antimicrobial compounds as an alternative to the conventional preservation techniques used in foods that are derived from fruit. For this purpose, a systematic review was carried out on the direct application of natural antimicrobials (free form) to fruit-derived products, and their use after covalent immobilization on the surface of different food-grade supports, to be employed as food preservatives or food processing aids to avoid modifying the sensory properties of the food. This review includes the advantages and disadvantages of utilizing natural antimicrobials according to their formulation (free or immobilized forms) and their application form on the food product (preservative or processing aid). A description of the main methodologies for the covalent immobilization of natural antimicrobials and the most relevant characterization techniques to verify covalent grafting and bioactivity is also included. Finally, future perspectives of these alternative preservation technologies are proposed.

A systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines [9] was conducted to compile the most relevant studies using natural antimicrobials in fruit-derived foods using two dosage forms: free (Section 3) or immobilized (Section 4). The selection process was carried out in different stages, as described below.

The selection process proposed by Bhardwaj et al. (2023) [10] and Rifat et al. (2022) [11] was followed for this stage. Three reviewers independently carried out the literature searches and checked the number of records. The employed research databases were Scopus, Web of Science, and PubMed. The search strategy was carried out by taking into account that “naturally-occurring antimicrobial compounds” covered the following terms: essential oils, fatty acids, lysozyme, chitosan, bacteriocin, bacteriophage. Subsequently, the following combined queries (“naturally-occurring antimicrobial compounds” AND “wine”) OR (“naturally-occurring antimicrobial compounds” AND “juice”) OR “naturally-occurring antimicrobial compounds” AND “jam”) OR (“naturally-occurring antimicrobial compounds” AND “beverage”) were used. Of all the found reports, all those whose publication range was between 2017 and 2023, and written in English, were selected.

The search results were exported to Mendeley reference manager and duplicates were removed.

Documents were screened according to the inclusion and exclusion criteria in the title and abstract. Eligible articles were evaluated by three reviewers after considering the inclusion and exclusion criteria for the full text (Table 1). Variations in the number or records in each stage were checked and any discrepancies were assessed by the reviewers to make a final decision. Information about the number of records included in this review and the summary of the selection process of the included articles are shown in Figure 1.

The systematic review on the use of natural antimicrobials in fruit-derived foods revealed that essential oil components (EOCs), bacteriocins, polysaccharides, organic acids, or bacteriophages are the most frequent molecules employed as preservatives in fruit-derived foods, probably due to their consideration as GRAS (generally recognized as safe) products by the U.S. FDA [12]. EOCs are a mixture of different compounds, such as terpenes, alcohols, phenols, etc., generated from plants. Free hydroxyl functional groups (-OH) are mostly responsible for their antimicrobial activity [13]. Bacteriocins are the peptides obtained from bacteria that are capable of changing the permeability of microorganisms by provoking their lysis. Of all the bacteriocins, nisin, iturin A, natamycin, bovicin, and thurincin H are proposed as antimicrobials [14,15,16,17,18]. The most important polysaccharide is chitosan, a biopolymer generated by the deacetylation processes of chitin. Its antimicrobial action is based mainly on the interaction between chitosan cationic groups and microorganisms [19]. Organic acids cover different compounds that present a deprotonated carboxyl group at a neutral pH. The interaction between the active group and a microorganism’s membrane is the main antimicrobial mechanism [20]. Bacteriophages are viruses that penetrate a specific bacterial host, spread within the host, and release more phages after cell lysis [21]. The antimicrobial activity of the naturally occurring compounds was tested in different fruit-derived products (i.e., wine, fruit juices, or soft drinks) against bacteria, such as Escherichia coli, Salmonella enterica, and Listeria monocytogenes, yeasts, such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, and Saccharomyces cerevisiae. In the reviewed studies, the authors reported remarkable efficacy for the microbiological control of all the fruit-derived foods. Table 2 summarizes the different studies that evaluated the antimicrobial efficacy of the aforementioned compounds in fruit-derived foods. Despite the demonstrated in vivo inhibitory efficacy of antimicrobials, some studies have revealed that their direct addition to food presents certain limitations that stem from their intrinsic physico-chemical properties. Mitropoulou et al. (2020) [22] and Thomas-Popo et al. (2019) [13] expressed that essential oils (EOs) and their components (EOCs) are poorly soluble in aqueous media and highly volatile when studied for antimicrobial purposes in fruit juices. Campion et al. (2017) [23] indicated that nisin is more stable at an acid pH when tested in milk and apple juice media. Liao et al. (2017) [15] reported that the temperature and salt concentration of media can affect nisin activity for apple juice stabilization. In addition to all these limitations, some reports have shown that the incorporation of these compounds modifies some food sensory attributes. The study of Beristaín-Bauza et al. (2018) [24] evaluated the sensory impact of 0–100 µg/mL of cinnamaldehyde and vanillin in coconut water. These authors reported the lowest general acceptability values at the highest concentration for both antimicrobial compounds. Chung et al. (2018) [25] tested the addition of thymol to different citrus extracts (lime, lemon, and calamarsi) and quantified a lower overall acceptability (p < 0.05) by incorporating thymol at concentrations up to 2 mM. Further research rated the appearance, odor, taste, aftertaste, viscosity, and overall acceptance parameters at different concentrations of isoeugenol (0–1 µL/mL) added to pineapple juice [13]. The results showed a significant decrease (p < 0.05) in the odor, taste, aftertaste, and overall acceptance scores, while no changes were recorded for the appearance and viscosity characteristics. Using EOs from Citrus medica and Cinnamomum zeylanicum for microbiological wine stabilization [22], aroma and taste assays were carried out. The results indicated that the incorporation of EOs significantly affected both parameters. Indeed, the product was rejected when concentrations over 0.010% of EOs were added because EOs masked the wine taste and also formed an oily layer on wine. The interaction of antimicrobial compounds with food matrix components also limits their application in food products [13,17,21,26,27,28,29,30]. Certain nutrients in food can have a protective effect on microorganisms; therefore, it would be necessary to use higher concentrations of natural antimicrobials. However, increasing the antimicrobial effective dose can result in more limitations that derive from applying a high concentration of certain natural antimicrobial compounds, as previously highlighted. For all these reasons, research is currently exploring new alternative dosage forms of natural antimicrobials to be used as food preservatives or processing aids, such as encapsulation of immobilization. The first alternative consists of trapping an active agent (i.e., a natural antimicrobial) in a carrier material to enhance its later release in the food or in the gastrointestinal tract. Immobilization, in contrast, consists of anchoring the active biomolecule on the surface of the support. This technology not only makes it possible to preserve the native antimicrobial properties of the active biomolecule but also prevents its leaching into the food matrix due to the creation of covalent bonds between the support and the antimicrobial compound. Having this in mind, covalent immobilization is presented in this work as an eco-friendly postharvest technology with great possibilities for application in the preservation of fruit-derived foods.

The use of immobilized natural antimicrobials to preserve or extend the shelf life of fruit-derived products has increased in recent years owing to their marked antimicrobial effectiveness and good capacity to cushion their sensory and stability impact on foods after grafting [48,49,50]. Immobilization refers to the chemical, physico-chemical, or electrostatic binding of bioactive molecules to a surface. Chemical immobilization involves the formation of at least one covalent bond between the surface and the target biomolecule, which represents the most permanent and irreversible form of coupling. Covalent linkage involves strongly bounding the compound of interest with a potentially longer shelf life, greater bioactivity, and lower toxicological risk [51]. The immobilization of natural antimicrobials on the surface of different materials is an approach that allows contact-killing materials to be obtained through antimicrobial molecules that are covalently attached to the surface. With this immobilization procedure, antimicrobials are exposed to the external environment, which enables direct contact between the immobilized molecule and the target microorganism [52]. According to this systematic review, these antimicrobial systems can be applied to fruit-derived foods in different processing stages: (i) as a food additive (preservative) present in the final product and (ii) as food processing aids that are absent in the final product (see Figure 2). This section focuses on discussing application cases of immobilized natural antimicrobials in fruit-derived products by differentiating these two application approaches.

In the last few years, the design of immobilized antimicrobial systems as food preservatives to control or prevent microbial spoilage in fruit-derived foods has grown. Table 3 summarizes the four studies found in the systematic review about immobilized natural antimicrobial compounds on food-grade supports to be used as food preservatives in fruit-derived products. For all the identified applications, besides the description of the support, immobilization technique, target microorganism, and the food matrix that is the study object, the physico-chemical and sensory impact after their incorporation into the matrix is discussed. In this context, Ribes et al. (2017) [50] conducted the first work, in which EOCs immobilized by an imine bond on the surface of silica supports were employed as promising antifungal agents to control strawberry jam decay without altering the final product’s sensory perception. Based on the marked antimicrobial activity of these promising preservatives, Ribes et al. (2019) [53] also investigated the synergistic effect of EOCs immobilized on the surface of silica particles against the bacteria and yeasts present in fruit juice and their influence on the food matrix. This work demonstrated the feasibility of combining immobilized antimicrobials to improve the microbial stabilization of fruit juice given that immobilization masks the undesirable aroma of EOCs in the food matrix according to both the gas chromatography and sensory evaluation results. Similarly, the antimicrobial activity of thymol immobilized on hollow mesoporous silica particles (HMSNs) in a real food system was investigated for the first time by Liu et al. (2022) [54]. It is noteworthy in this study that the antimicrobial agent was covalently grafted to the silica support by running a reaction with 3-(triethoxysilyl)-propyl isocyanate, which resulted in carbamate bonding instead of the imine bonding described in the above-mentioned examples. The EOC immobilized on HMSNs showed an excellent potential for enhancing the antimicrobial activity of thymol against foodborne bacteria. It also reduced the impact of EOCs on the final product’s physico-chemical properties, such as color, pH, and soluble solids content. However, the impacts on the most relevant organoleptic properties (aroma and flavor) were not evaluated. Recently, different antimicrobial systems based on the covalent immobilization of chitosan for the microbial control of apple juice were developed by Ruiz-Rico et al. (2023) [55]. The use of chitosan-coated supports as food preservatives in juice reduced the food matrix’s microbial load, which increased its shelf life, although the impact on juice was not evaluated. All these examples confirm the potential of antimicrobial systems based on immobilized natural antimicrobials as food preservatives in the food industry after regulatory authorities have approved them. Finally, it is worth mentioning that all these reviewed systems present common characteristics: (i) the bioactive compound is covalently immobilized on the surface of the support; (ii) immobilized bioactive compounds exhibit greater antimicrobial or antifungal activity than their free form; (iii) the use of these novel preservatives does not modify the final product’s physico-chemical or sensory properties.

The second approach of using immobilized natural antimicrobials in fruit-derived foods does not imply the permanence of particles in food to exert their antimicrobial effect. So in this case, instead of considering the antimicrobial supports to be food preservatives, they should be taken as processing aids. In the systematic review of this application, four works used immobilized antimicrobials as processing aids. Within this framework, Song et al. (2019) [56] employed iron oxide nanoparticle–polydopamine–nisin composites with magnetic characteristics to control A. acidoterrestris growth in apple juice and recovered particles after treatment. After demonstrating that the juices treated with functionalized magnetic particles were not influenced in physico-chemical and sensory terms, as well as the non-toxicity and biosecurity of composites, the authors highlighted this innovative antimicrobial system as a promising tool to control A. acidoterrestris contamination in the juice industry. Conversely, the other three studies applied the immobilization of natural antimicrobial compounds on food-grade supports as a strategy to create filtration systems for the cold pasteurization of liquid fruit-derived foods. The main objective of these filtration systems was to remove spoilage or pathogenic microorganisms from liquid fruit-derived products (juices or wine) through the retention and/or the disruption of the bacterial cell wall after the interaction with the antimicrobial compound [57]. Zhang et al. (2021) [58] reported employing nisin-coated polyvinylidene difluoride microfiltration membranes (pore diameter of 0.22 μm) to eliminate A. acidoterrestris contamination from apple juice due to the antibacterial action of nisin and the retention of spores on the membrane surface. In a different approach, Peña-Gomez et al. (2019) [49] developed novel filtering materials based on silica microparticles (50 μm) functionalized with EOCs as an alternative cold pasteurization method for apple juice by depth filtration. In a first assay, the developed filtration system was able to reduce the E. coli load inoculated in pasteurized apple juice of at least 5 logarithmic reduction values (LRVs). In addition, employing antimicrobial particles for the filtration of fresh juices was able to microbiologically stabilize the non-thermally treated apple juice, which resulted in juice with high microbial stability and quality. This suggests that this filtration technology is a promising alternative to existing pasteurization technologies that apply heat. In another work, Ruiz-Rico et al. (2021) [59] evaluated filter aids based on the covalent immobilization of different EOCs and other phenolic compounds on wine microbiological stabilization. These filtering aids presented some advantages over standard filtration materials, such as minimal impact on wine sensory characteristics and high removal capacity. Likewise, they could be used for clarification, microbiological stabilization, and sterile filtration in a single continuous treatment, thus reducing wine losses and energy costs by compiling different traditional filtration stages in a single step, as well as enhancing the treatment’s overall hygiene and security. However, the authors pointed out the need to improve the stability of grafting and the reuse conditions or filter life before being applied in the food industry. More information about the specific natural antimicrobials, supports, immobilization techniques, and target microorganisms studied in the described examples can be found in Table 4. These described filtration systems differ from micro- and ultrafiltration techniques, which have been extensively studied and implemented for the cold pasteurization of drinks on an industrial scale, in terms of removal capacity and their impact on the properties of filtered drinks. The mechanism of action of micro- and ultrafiltration (pore size from 0.001 μm to <0.1 μm for ultrafiltration, and from 0.1 μm to 10 μm for microfiltration) is the physical retention of all the molecules and organisms bigger than the pore size. In contrast, filtration systems based on membranes or particles functionalized with natural antimicrobials exhibit a larger pore size or filtration channels that only allow the partial retention of microorganisms and food matrix components. Therefore, they act by the combined effect of cell retention and cell damage due to the specific interaction with the antimicrobial compound by preserving the nutritional, functional, and sensory properties of the filtered drink.

After identifying and analyzing the different examples of applying immobilized natural antimicrobials, this section describes the immobilization approaches in detail to provide a guideline of the synthesis and characterization of these antimicrobial systems for future developments. Chemical immobilization can be a complex process for preserving the antimicrobial properties of the target molecule on a specific surface. The specific properties of the substrate surface, such as composition, charge, hydrophilicity/hydrophobicity, chemical stability, roughness, and geometry, as well as antimicrobial characteristics, including molecular structure, charge, and molecular size, should be considered for the bonding process. In addition, covalent immobilization can alter the conformational molecule structure by altering its mechanism of action. Therefore, coupling strategies should be carefully evaluated to optimize the attachment and maintenance of antimicrobial properties [51].

The substrate surfaces for the immobilization of biomolecules are diverse in terms of their features and properties, but the main characteristic they must present for coupling is the presence of sufficient functional groups for attaching the target molecule [60]. Otherwise, it may be necessary to modify its surface for immobilization [61]. Of the food-grade materials permitted to come into contact with food, this review identified different organic and inorganic substrates. Organic materials, such as cellulose, chitosan, or synthetic polymers (i.e., polystyrene, polyethylene, polyamide, or polyvinylidene difluoride), are commonly used substrates for biomolecule immobilization. The chitosan structure presents many hydroxyl and amine groups that enable the effective grafting of biomolecules without involving any modification [62]. Other organic substrates present low or non-reactive functional moieties that require previous surface activation for specific grafting reactions [63]. Of the available inorganic substrates, ceramic materials such as silica, clay, sand, or glass, and metallic materials such as iron oxide, zinc oxide, titanium dioxide, or stainless steel are suitable support materials for biomolecule immobilization. Silica is the most widely used inorganic support material for biomolecule immobilization, which can be obtained with diverse structures such as highly ordered crystalline forms, non-periodic porous systems, mesoporous amorphous solids (i.e., M41S or SBA-n family supports), non-porous amorphous forms, or totally random structures [60,64]. The natural presence of functional moieties (hydroxyl groups) on the surface of some ceramic materials, such as silica, or the surface preactivation required for other inert surfaces, such as glass or stainless steel, allow the covalent binding of biomolecules [62].

Surface activation is a set of methods used to alter the chemistry of a substrate surface by introducing chemical groups or charges on the surface. Different techniques are available to make surface modifications to supports prior to immobilization, including silanization as the main strategy [61]. Silanization involves the covering of a substrate surface rich in hydroxyl groups with organo-functional alkoxysilane moieties. Silane-coupling agents, of which 3-aminopropyltriethoxysilane (APTES) is the most representative organosilane, present an alkoxy group and an organofunctional group (amine, thiol, isocyanate, or carboxyl moieties). The alkoxy moiety forms hydrogen bonds with hydroxide groups of the substrate surface, while the organofunctional moiety enables the immobilization of biomolecules that display reactive functionalities, such as amines, carboxylic acids, or aldehydes [65]. With silica supports, the silanization procedure can be applied during the synthesis of supports (co-condensation functionalization) or after preparing the support (post-synthesis grafting) [64]. Another strategy to modify the surface reactivity of substrates is biomimetic coating with dopamine. This coating simulates the adhesive properties of marine adhesives by using dopamine that adheres and polymerizes on surfaces, such as metal oxides or polymeric surfaces, although the binding mechanism is not well established [66]. Once the substrate surface has been activated, biomolecule immobilization on the material’s surface can take place.

For covalent immobilization purposes, the functional moieties of the biomolecule must be compatible with the reactive groups present, either spontaneously or occurring after a previous surface modification on the substrate surface. The biomolecule chemical structure should be studied to preserve antimicrobial properties. The main drawback of covalent immobilization is the potential disruption of antimicrobial activity after bond formation. Immobilized biomolecules can be attached to the surface by a specific site that is responsible for the inhibitory potential, or in a rigid spatial orientation that can significantly change antimicrobial properties. In addition, the grafting process should ensure suitable coating density and uniformity to guarantee the reproducibility and scale up of immobilization, as well as the preservation of the grafted biomolecule’s efficient antimicrobial activity [52]. Therefore, the bonding approach should be carefully designed. Biomolecules that contain intrinsic chemically reactive groups, such as carboxylic acid, amino, sulfhydryl, and hydroxyl groups, can be covalently immobilized onto activated surfaces through chemical interfacial reactions by non-selective immobilization. This type of grafting can result in more than one type of covalent bonding with different biomolecule orientations [51]. In contrast, biomolecules that do not possess convenient chemical groups should be modified by diverse chemical strategies. To convert one functional group into another or to assist the grafting process, cross-linking agents, such as glutaraldehyde, carbodiimides, etc., can be used to obtain the needed functional groups, such as thiols, aldehydes, carboxylic acids, hydroxyls, and primary amines [61]. The insertion of a specific functional group allows a selective covalent bond to be achieved between the biomolecule and the substrate with a specific conformation [51].

The aim of antimicrobial immobilization approaches is to design antimicrobial-coated substrates that should exert their biocidal properties after grafting without apparent release, and result in increased stability, a longer shelf life, and enhanced bioactivity [48]. To ensure that this goal is fulfilled, a detailed characterization of the developed antimicrobial-coated materials is important to verify the biomolecule’s covalent immobilization and to help the reliable interpretation of its antimicrobial mechanism of action after grafting. Surface chemistry should be characterized by analytical techniques to establish the coating’s coverage and the effect of coating on the substrate microstructure, surface charge, surface morphology, porosity, and size [67]. These techniques include thermal analyses (i.e., thermogravimetric analysis and differential scanning calorimetry), spectroscopic techniques (i.e., infrared spectroscopy, X-ray spectroscopy, and nuclear magnetic resonance spectroscopy), microscopic techniques (i.e., electron microscopy and scanning microscopy), and other instrumental analyses (i.e., elemental analysis, laser scattering or diffraction analysis, and zeta potential analysis). The biocidal properties of the immobilized antimicrobial are characterized by in vitro antimicrobial performance testing against microorganisms of interest in different microbial life stages [68]. These viability tests can be combined with microscopic and molecular techniques to reduce errors from viable but non-culturable (VBNC) microorganisms and to help to elucidate the mechanism of action of the grafted antimicrobial [69]. In situ antimicrobial testing is also needed to evaluate biocidal properties in real food matrices, the impact of the immobilized antimicrobial on food properties, and the potential leaching of the grafted biomolecule [70]. The simulation of the other parameters relevant to the real application of the developed immobilized antimicrobials is another important factor for characterization. Shelf life, cleaning, and stability requirements should be characterized to establish adequate durability [67]. Prior to a real application, the biocompatibility and safety of the immobilized antimicrobials should be characterized by toxicity studies that employ relevant human cell lines and animal models [71,72].

Seeking new technologies capable of preserving fruit-derived foods and minimizing the impact of processing on the sensory, nutritional, and functional properties of fruit-derived foods, while reducing the carbon footprint of thermal processing, are major challenges for today’s food industry. With such tasks, the solution could come from a new approach to the application of natural antimicrobials: their immobilization on food-grade supports. The immobilization of different natural antimicrobials (mostly EOCs) on silica particles is a proven excellent strategy to create effective food preservatives (direct use) or processing aids (indirect use) capable of improving the microbiological quality of fruit-derived foods by extending their shelf life, but without altering nutritional, functional, and sensory properties. Beyond these advantages, this alternative approach to traditional heat treatment is also seen as an eco-friendly postharvest technology. On the one hand, it could contribute to improving the decarbonization and energy efficiency of the fruit-derived processing sector because it avoids using heat. On the other hand, it provides solutions based on nature, such as natural antimicrobial biomolecules. Moreover, as most of these proposed natural antimicrobials are obtained from plant and animal by-products, their valorization will comply with bioeconomy principles by reducing waste and generating added value to the agri-food sector. Finally, this technology might be used to preserve fruit-derived foods in developing countries with restricted access to other complex and expensive technologies or electricity. As limitations to the immediate use of this technology, it should be noted that all the applications shown in this work have been exclusively investigated on a laboratory scale, and the immobilization processes described in the different works are laborious and require energy and solvents. These limitations could be reduced by employing green chemistry to reduce the use of solvents and photocatalysis by resorting to solar or white light as a source of energy for the immobilization steps. In either case, this review demonstrates how the immobilization of natural antimicrobial compounds on food-grade supports possesses all the features required to be proposed as an eco-friendly postharvest technology that is capable of preserving fruit-derived foods.