Characterization of antimicrobial multilayer film based on ethylcellulose-pectin incorporated with nanoemulsions of trans-cinnamaldehyde essential oil.

In this study, polymer solution casting was utilized to fabricate a multilayer film with ethylcellulose (EC) as the outer layers and trans-cinnamaldehyde-loaded pectin as the inner layer. A significant increase in whiteness and UV-visible light blocking capability and a remarkable decrease in total color difference and yellowness of the films were seen via increasing the thickness of EC outer layers. Scanning electronic microscopy observation showed that the inner and outer layers had a smooth and uniform surfaces with clear boundary. The thicker film has better stretchability and strength, but is less flexible than thinner film. Glass transition temperature did not change remarkably with increasing thickness of EC outer layers, but thermal stability was slightly improved. FTIR-ATR spectra revealed the formation of hydrogen bonds between the two adjacent layers. The multilayer films exhibited excellent antimicrobial efficacy against Gram-positive and Gram-negative foodborne pathogens. The results suggested that this multilayer film has potential applications in active food packaging.

The impact of different acidic conditions and food substrates on Listeria monocytogenes biofilms development and removal using nanoencapsulated carvacrol.

Listeria monocytogenes biofilms present a significant challenge in the food industry. This study explores the impact of different acidic conditions of culture media and food matrices on the development and removal of biofilms developed on stainless steel surfaces by wild-type (WT) L. monocytogenes strains as well as in two mutant derivatives, ΔsigB and ΔagrA, that have defects in the general stress response and quorum sensing, respectively. Additionally, the study investigates the efficacy of nanoencapsulated carvacrol as an antimicrobial against L. monocytogenes biofilms developed in Tryptic Soy Broth (TSB) culture media acidified to different pH conditions (3.5, 4.5, 5.5, 6.5), and in food substrates (apple juice, strained yogurt, vegetable soup, semi-skimmed milk) having the same pH levels. No biofilm formation was observed for all L. monocytogenes strains at pH levels of 3.5 and 4.5 in both culture media and food substrates. However, at pH 5.5 and 6.5, increased biofilm levels were observed in both the culture media and food substrates, with the WT strain showing significantly higher biofilm formation (3.04-6.05 log CFU cm-2) than the mutant strains (2.30-5.48 log CFU cm-2). For both applications, the nanoencapsulated carvacrol demonstrated more potent antimicrobial activity against biofilms developed at pH 5.5 with 2.23 to 3.61 log reductions, compared to 1.58-2.95 log reductions at pH 6.5, with mutants being more vulnerable in acidic environments. In food substrates, nanoencapsulated carvacrol induced lower log reductions (1.58-2.90) than the ones in TSB (2.02-3.61). These findings provide valuable insights into the impact of different acidic conditions on the development of L. monocytogenes biofilms on stainless steel surfaces and the potential application of nanoencapsulated carvacrol as a biofilm control agent in food processing environments.

Microencapsulation via Spray-Drying of Geraniol-Loaded Emulsions Stabilized by Marine Exopolysaccharide for Enhanced Antimicrobial Activity.

The current study investigates the formation of microencapsulated geraniol powder, with the exopolysaccharide EPS-K1B3 produced by Halomonas caseinilytica K1, as wall material, using spray-drying. Evaluation of the antimicrobial activity of the functional emulsions, prepared at either pH 5 or pH 7, was carried out against Gram-positive (Listeria innocua (ATCC 33090)) and Gram-negative (Escherichia coli (DSM682)) bacterial strains. Results showed prolonged antimicrobial efficacy until 30 days of incubation for geraniol microcapsules compared to wet geraniol emulsions, which could confirm the ability of the spray-drying process to protect encapsulated geraniol for a longer period. The highest antimicrobial efficacy of geraniol microcapsules was observed against L. innocua at pH 5. Therefore, the influence of pH on the functional property of geraniol microcapsules could be highlighted beside the targeted bacterial strain.

Nanoemulsion-Based Multilayer Films for Ground Beef Preservation: Antimicrobial Activity and Physicochemical Properties.

This study aimed to improve the physical, mechanical, and biological properties of a monolayer pectin (P) film containing nanoemulsified trans-Cinnamaldehyde (TC) by incorporating it between inner and outer layers of ethylcellulose (EC). The nanoemulsion had an average size of 103.93 nm and a zeta potential of -46 mV. The addition of the nanoemulsion increased the opacity of the film, reduced its moisture absorption capacity, and improved its antimicrobial activity. However, the tensile strength and elongation at break of the pectin films decreased after the incorporation of nanoemulsions. Multilayer films (EC/P/EC) showed a higher resistance to breaking and better extensibility compared to monolayer films. The antimicrobial activity of both mono and multilayer films was effective in inhibiting the growth of foodborne bacteria during storage of ground beef patties at 8 °C for 10 days. This study suggests that biodegradable antimicrobial multilayer packaging films can be effectively designed and applied in the food packaging industry.

Physicochemical and Biological Characterization of Encapsulated Olive Leaf Extracts for Food Preservation.

Phenolic compounds in olive leaves have an excellent antioxidant activity and good antimicrobial properties. These bioactive molecules have beneficial properties for health, arousing great scientific and commercial interest. This study reports lyophilized olive leaf extracts (OLE) encapsulated by spray-drying using maltodextrins, maltodextrins-pectin and maltodextrins-gum Arabic as encapsulating agents. Lyophilized OLE were collected from two varieties cultivated in a harsh pedo-climatic conditions of the arid region of Tunisia. The effects of the genetic factor and the different encapsulating agents on the physicochemical properties of microcapsules and their behavior during storage, as well as their antimicrobial activities, were studied. Microcapsules successfully passed heat treatment and storage conditions and their antimicrobial activities were preserved. The encapsulating agent combination improved the encapsulation efficiency and the product yield in Zarrazi variety compared to Dhokar one. In addition, Dhokar variety microparticles showed the best heat stability at 4 and 25 °C after 90 days of storage and the higher inhibition percent against bacteria. The results of the present study evidenced that the best conditions for OLE encapsulation were obtained when the maltodextrins-pectin and maltodextrins-gum Arabic were combined to form a hybrid coating material.

Pepsin and Trypsin Treatment Combined with Carvacrol: An Efficient Strategy to Fight Pseudomonas aeruginosa and Enterococcus faecalis Biofilms.

Biofilms consist of microbial communities enclosed in a self-produced extracellular matrix which is mainly responsible of biofilm virulence. Targeting this matrix could be an effective strategy to control biofilms. In this work, we examined the efficacy of two proteolytic enzymes, pepsin and trypsin, to degrade P. aeruginosa and E. faecalis biofilms and their synergistic effect when combined with carvacrol. The minimum dispersive concentrations (MDCs) and the contact times of enzymes, as well as the minimal inhibitory concentrations (MICs) and contact times of carvacrol, were determined against biofilms grown on polystyrene surfaces. For biofilms grown on stainless steel surfaces, the combined pepsin or trypsin with carvacrol treatment showed more significant reduction of both biofilms compared with carvacrol treatment alone. This reduction was more substantial after sequential treatment of both enzymes, followed by carvacrol with the greatest reduction of 4.7 log CFU mL−1 (p < 0.05) for P. aeruginosa biofilm and 3.3 log CFU mL−1 (p < 0.05) for E. faecalis biofilm. Such improved efficiency was also obvious in the epifluorescence microscopy analysis. These findings demonstrate that the combined effect of the protease-dispersing activity and the carvacrol antimicrobial activity could be a prospective approach for controlling P. aeruginosa and E. faecalis biofilms.

Effect of trans-cinnamaldehyde or citral on sodium caseinate: Interfacial rheology and fluorescence quenching properties.

The interactions between sodium caseinate (CAS) and two natural aldehydes (trans-cinnamaldehyde (TC) and citral) were studied by evaluating oil/water (O/W) interfacial and fluorescence quenching properties. A small amount of TC in the oily phase resulted in lower O/W interfacial tension (9.12 mN/m). Particularly, the use of TC developed a stronger interface with higher elastic moduli (∼16.21 mN/m). This was supported by the fluorescence measurements: the quenching effect of TC on CAS was more pronounced than that of citral. Kinetic analysis indicated that both dynamic and static quenching occurs. The large binding constant (1.78 × 105 M-1) at 25 °C suggests that TC has strong affinity for CAS. Meanwhile, this binding process seemed to be spontaneous and driven by hydrogen bond formation with unfavorable conformational changes. This work would provide guidance for using the binding properties of natural aldehydes to enhance the interfacial properties of proteins.

Dynamic Salmonella Enteritidis biofilms development under different flow conditions and their removal using nanoencapsulated thymol.

In food industries, microbial contaminations are difficult to control due to the recurrent formation of biofilms that hinders antimicrobials penetration and efficiency. An understanding of Salmonella Enteritidis biofilms behavior under flow conditions is a key to develop efficient preventive and control strategies. S. Enteritidis biofilms displayed 5.96, 6.28 and 6.80 log CFU cm-2 under 0.006 cm s-1, 0.045 cm s-1, and 0.087 cm s-1 flow velocities, respectively. Biofilms exposed to higher nutrient conditions under greater flow rates, induced significantly more biofilm biomass. To control biofilms, the disinfection efficiency of thymol (THY) was assessed under dynamic conditions by encapsulation it into two types of nanocapsules: monolayer (ML) nanocapsules prepared with a single carrier material (maltodextrin), and layer-by-layer (LBL) nanocapsules prepared by combining two carrier materials (maltodextrin and pectin). A combined mixture of ML and LBL nanocapsules at ½ their minimal inhibitory concentrations induced 99.99% eradication of biofilms developed under the highest flow conditions, after 5 h. ML nanocapsules decreased significantly bacterial counts during the first 0.5 h, while LBL nanocapsules eliminated the remaining bacterial cells and ensured a protection from bacterial contamination for up to 5 h by releasing THY in a sustained manner over time due to the thicker shell wall structure.

Hurdle technology based on the use of microencapsulated pepsin, trypsin and carvacrol to eradicate Pseudomonas aeruginosa and Enterococcus faecalis biofilms.

The biofilm lifestyle plays a major role in the resistance and virulence of Pseudomonas aeruginosa and Enterococcus faecalis. In this study, two microencapsulated proteases (pepsin ME-PEP and trypsin ME-TRYP) were evaluated for their biofilm dispersal activity and their synergistic effect with microencapsulated carvacrol (ME-CARV). Spray-drying was used to protect enzymes and essential oil and enhance their activities. Cell count analysis proved the synergistic activity of enzymes and carvacrol treatment as biofilms were further reduced after combined treatment in comparison to ME-CARV or enzymes alone. Furthermore, results showed that sequential treatment in the order ME-TRYP - ME-PEP - ME-CARV resulted in more efficient biofilm removal with a maximum reduction of 5 log CFU mL-1 for P. aeruginosa and 4 log CFU mL-1 for E. faecalis. This study proposes that the combination of microencapsulated proteases with ME-CARV could be useful for the effective control of P. aeruginosa and E. faecalis biofilms.

Understanding of the key factors influencing the properties of emulsions stabilized by sodium caseinate.

Emulsions can be easily destabilized under various conditions during preparation and storage. Therefore, it is necessary to understand the factors that influence the stability of emulsions, which is essential for their subsequent studies. Sodium caseinate (CAS) is a well-used nutritional and functional ingredient in emulsion preparation due to its good solubility and emulsifying properties. CAS-stabilized emulsions can be considered good food emulsion delivery systems, but their applications are still limited under certain conditions due to their instability to creaming and aggregation. Therefore, the purpose of this review is to provide a complete overview of how different environmental stresses and processing conditions affect the stability of CAS-stabilized emulsions and how to improve their stability. Initially, the general properties of CAS as emulsifiers and the characterization of CAS-stabilized oil-in-water (O/W) emulsions were summarized. Second, the major instability mechanisms that operate in CAS-stabilized emulsions were presented. Furthermore, the general factors such as pH, emulsifier concentration, ionic strength, oxidation, and processing conditions, affecting the stability of CAS-stabilized O/W emulsion, were discussed. On this basis, the commonly used methods for evaluating emulsion stability are introduced. Finally, state-of-the-art strategies to improve CAS-based emulsion stability are also described and summarized. This review is expected to provide a theoretical basis for the future applications of CAS in food emulsions.

Microencapsulation of carvacrol as an efficient tool to fight Pseudomonas aeruginosa and Enterococcus faecalis biofilms.

Biofilms are involved in serious problems in medical and food sectors due to their contribution to numerous severe chronic infections and foodborne diseases. The high resistance of biofilms to antimicrobial agents makes their removal as a big challenge. In this study, spray-drying was used to develop microcapsules containing carvacrol, a natural antimicrobial agent, to enhance its activity against P. aeruginosa and E. faecalis biofilms. The physicochemical properties and microscopic morphology of the realized capsules and cells were characterized. The minimum inhibitory concentration of encapsulated carvacrol (E-CARV) (1.25 mg mL-1) was 4-times lower than that of free carvacrol (F-CARV) (5 mg mL-1) against P. aeruginosa, while it remained the same against E. faecalis (0.625 mg mL-1). E-CARV was able to reduce biofilm below the detection limit for P. aeruginosa and by 5.5 log CFU ml-1 for E. faecalis after 15 min of treatment. Results also showed that F-CARV and E-CARV destabilize the bacterial cell membrane leading to cell death. These results indicate that carvacrol exhibited a strong antimicrobial effect against both bacterial biofilms. In addition, spray-drying could be used as an effective tool to enhance the antibiofilm activity of carvacrol, while reducing the concentrations required for disinfection of abiotic surfaces.

Formation of lysozyme-caseinate heteroprotein complexes for encapsulation of lysozyme by spray-drying: Effect of mass ratio and temperature.

The formation of heteroprotein complexes obtained by the interactions between sodium caseinate (CAS) and lysozyme (LYS) at pH 7 was investigated by using turbidimetric analysis, particle size distribution, and zeta potential at different CAS/LYS ratios. Moreover, isothermal titration calorimetry (ITC) was used to determine the type and magnitude of the energies involved in the CAS/LYS complexation process and evaluated the thermodynamic behavior of their complexation. Results revealed that the structure of CAS/LYS complexes drastically changed when CAS/LYS ratio increased to 1.0 and the structuring stages were characterized by exothermic signals and were controlled by favorable enthalpy changes due to electrostatic interactions between both proteins. In addition, the interaction between two proteins was temperature-dependent and mainly entropy-driven, which was verified by molecular dynamics (MD) simulations, and the hydrophobic interactions and hydrogen bonding were shown to play an important role in CAS/LYS interactions. Furthermore, CAS/LYS complexes showed minimum LYS enzymatic activity at CAS/LYS ratio 1.0. Though spray-drying of CAS/LYS complexes with ratio 1.0, the LYS activity in reconstituted solution was recovered >80 % of initial activity after calcium chloride addition. The present study provides useful information about CAS/LYS complexation and binding processes, which could facilitate their application in antimicrobial edible food packaging.

Effect of pectin on the properties of nanoemulsions stabilized by sodium caseinate at neutral pH.

The effect of different concentrations of low methoxyl pectin (LMP) on lipid oxidation and physical stability of sodium caseinate (CAS) stabilized nanoemulsions under neutral pH was investigated. The addition of pectin at low concentration (≤ 0.10 wt%) had no significant effect on the average size of nanoemulsions, but a slight size increase and phase separation were observed at higher concentrations of pectin (≥ 0.25 wt%). This result suggests that LMP can not adsorb at the oil/water interfacial CAS membrane at neutral pH. However, in the presence of LMP, the physical stability of nanoemulsions against high salt concentrations and freeze-thaw cycles was significantly enhanced. Moreover, nanoemulsions containing pectin have a better ability to inhibit lipid and protein oxidation than nanoemulsions without pectin after 3 weeks, and the lowest lipid hydroperoxide content was observed for nanoemulsions containing 0.25 wt% pectin.

Advancements in Biodegradable Active Films for Food Packaging: Effects of Nano/Microcapsule Incorporation.

Food packaging plays a fundamental role in the modern food industry as a main process to preserve the quality of food products from manufacture to consumption. New food packaging technologies are being developed that are formulated with natural compounds by substituting synthetic/chemical antimicrobial and antioxidant agents to fulfill consumers' expectations for healthy food. The strategy of incorporating natural antimicrobial compounds into food packaging structures is a recent and promising technology to reach this goal. Concepts such as "biodegradable packaging", "active packaging", and "bioactive packaging" currently guide the research and development of food packaging. However, the use of natural compounds faces some challenges, including weak stability and sensitivity to processing and storage conditions. The nano/microencapsulation of these bioactive compounds enhances their stability and controls their release. In addition, biodegradable packaging materials are gaining great attention in the face of ever-growing environmental concerns about plastic pollution. They are a sustainable, environmentally friendly, and cost-effective alternative to conventional plastic packaging materials. Ultimately, a combined formulation of nano/microencapsulated antimicrobial and antioxidant natural molecules, incorporated into a biodegradable food packaging system, offers many benefits by preventing food spoilage, extending the shelf life of food, reducing plastic and food waste, and preserving the freshness and quality of food. The main objective of this review is to illustrate the latest advances in the principal biodegradable materials used in the development of active antimicrobial and antioxidant packaging systems, as well as the most common nano/microencapsulated active natural agents incorporated into these food-packaging materials.

Hurdle technology using encapsulated enzymes and essential oils to fight bacterial biofilms.

Biofilm formation on abiotic surfaces has become a major public health concern because of the serious problems they can cause in various fields. Biofilm cells are extremely resistant to stressful conditions, because of their complex structure impedes antimicrobial penetration to deep-seated cells. The increased resistance of biofilm to currently applied control strategies underscores the urgent need for new alternative and/or supplemental eradication approaches. The combination of two or more methods, known as Hurdle technology, offers an excellent option for the highly effective control of biofilms. In this perspective, the use of functional enzymes combined with biosourced antimicrobial such as essential oil (EO) is a promising alternative anti-biofilm approach. However, these natural antibiofilm agents can be damaged by severe environmental conditions and lose their activity. The microencapsulation of enzymes and EOs is a promising new technology for enhancing their stability and improving their biological activity. This review article highlights the problems related to biofilm in various fields, and the use of encapsulated enzymes with essential oils as antibiofilm agents. KEY POINTS: • Problems associated with biofilms in the food and medical sectors and their subsequent risks on health and food quality. • Hurdle technology using enzymes and essential oils is a promising strategy for an efficient biofilms control. • The microencapsulation of enzymes and essential oils ensures their stability and improves their biological activities.

Polyelectrolytes-stabilized liposomes for efficient encapsulation of Lactobacillus rhamnosus and improvement of its survivability under adverse conditions.

To improve the survivability of Lactobacillus rhamnosus probiotics, nanoliposomes (NLs) coated with chitosan (CH)-gelatin (GE) polyelectrolytes have been synthesized and characterized. The produced CH-GE-coated NLs containing L. rhamnosus had mean sizes in the range of 134.8-495.8 nm. HRTEM showed the smooth spherical shape of the vesicles. ATR-FTIR findings indicated the successful coating of the produced NLs by the used CH-GE polyelectrolytes. According to DSC results, CH-GE polyelectrolytes desorption on the surface of NLs altered the physical characteristics of the phospholipid bilayers. Here, an increase in the melting temperature (Tm) from 119.9 to 127.5 °C in L. rhamnosus-loaded CH-GE-coated NLs made this system more stable than uncoated liposomes. Furthermore, the CH-GE coated nanoparticles loaded with L. rhamnosus exhibited a significant enhancement in the viability of cells under simulated gastrointestinal fluids (SGF/SIF). These results may guide the potential application of polyelectrolytes-coated NLs as a carrier of probiotic cells in functional food development.

Essential oils and their active components applied as: free, encapsulated and in hurdle technology to fight microbial contaminations. A review.

Microbial contaminations are responsible for many chronic, healthcare, persistent microbial infections and illnesses in the food sector, therefore their control is an important public health challenge. Over the past few years, essential oils (EOs) have emerged as interesting alternatives to synthetic antimicrobials as they are biodegradable, extracted from natural sources and potent antimicrobials. Through their multiple mechanisms of actions and target sites, no microbial resistance has been developed against them till present. Although extensive documentation has been reported on the antimicrobial activity of EOs, comparisons between the use of whole EOs or their active components alone for an antimicrobial treatment are less abundant. It is also essential to have a good knowledge about EOs to be used as alternatives to the conventional antimicrobial products such as chemical disinfectants. Moreover, it is important to focus not only on planktonic vegetative microorganisms, but to study also the effect on more resistant forms like spores and biofilms. The present article reviews the current knowledge on the mechanisms of antimicrobial activities of EOs and their active components on microorganisms in different forms. Additionally, in this review, the ultimate advantages of encapsulating EOs or combining them with other hurdles for enhanced antimicrobial treatments are discussed.

Water-Soluble Ruthenium (II) Complex Derived From Optically Pure Limonene and Its Microencapsulation Are Efficient Tools Against Bacterial Food Pathogen Biofilms: Escherichia coli, Staphylococcus aureus, Enteroccocus faecalis, and Listeria monocytogenes.

Bioactive aminooxime ligands based on optically pure (R)-limonene have been synthesized in two steps. Their ruthenium (II) cationic water-soluble complex was prepared by a reaction between dichloro (para-cymene) ruthenium (II) dimers and aminooxime ligands in a 1:2 molar ratio. Antibacterial and antibiofilm activities of the synthetized complex were assessed against Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis. The results revealed that the ruthenium (II) complex has higher antibacterial and antibiofilm activities in comparison with free ligands or the enantiopure (R)-limonene. Moreover, microencapsulation of this complex reduced its cytotoxicity and improved their minimum inhibitory concentration and antibiofilm activity toward the considered bacteria. The ruthenium (II) complex targets the bacterial cell membrane, which leads to rapid leakage of intracellular potassium. Our study suggests that the developed ruthenium (II) complexes could be useful as an alternative to conventional disinfectants.

Nanocomposite sponges for enhancing intestinal residence time following oral administration.

In this work, nanocomposites that combine mucopenetrating and mucoadhesive properties in a single system are proposed as innovative strategy to increase drug residence time in the intestine following oral administration. To this aim, novel mucoadhesive chitosan (CH) sponges loaded with mucopenetrating nanoemulsions (NE) were developed via freeze-casting technique. The NE mucopenetration ability was determined studying the surface affinity and thermodynamic binding of the nanosystem with mucins. The ability of nanoparticles to penetrate across a preformed mucins layer was validated by 3D-time laps Confocal Laser Scanning Microscopy imaging. Microscopy observations (Scanning Electron Microscopy and Optical Microscopy) showed that NE participated in the structure of the sponge affecting its stability and in vitro release kinetics. When incubated with HCT 116 and Caco-2 cell lines, the NE proved to be cytocompatible over a wide concentration range. Finally, the in vivo biodistribution of the nanocomposite was evaluated after oral gavage in healthy mice. The intestinal retention of NE was highly enhanced when loaded in the sponge compared to the NE suspension. Overall, our results demonstrated that the developed nanocomposite sponge is a promising system for sustained drug intestinal delivery.

Preparation and Characterization of Microcapsules Containing Antioxidant Fish Protein Hydrolysates: a New Use of Bycatch in Brazil.

In this study, we evaluated the effect of complexation and microencapsulation with pea protein on the antioxidant activity of protein hydrolysates from bycatch in Brazil. The zeta potential values of complexes changed from negative to positive with the increase of pea protein as a result of positively charged complexes formation. The increase in the ratio of pea protein/hydrolysates also resulted in increased turbidity in all samples. Particle size measurements indicated that the complexes tended to form larger aggregates (ranged from 61.5 ± 1.7 µm to 183 ± 2.8 µm). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the pea protein/fish protein hydrolysate complexes was higher than that of the protein hydrolysates alone. Moreover, increasing levels of pea protein did not affect the antioxidant activity of fish protein hydrolysates. The complexes of the Paralonchurus brasiliensis were chosen for the microencapsulation process by spray-drying. The results revealed that spray-drying did not have a significant effect (P > 0.05) on the protein hydrolysate antioxidant activity when they were complexed with pea protein. Thus, this work suggests that the complexation with pea protein and subsequent microencapsulation by spray-drying is an efficient way to protect the biological activity of protein hydrolysates obtained from bycatch. This study provides evidence for the potential use of bycatch from shrimp fisheries as functional ingredients or nutraceuticals.