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.

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.

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.

Conditions of nisin production by Lactococcus lactis subsp. lactis and its main uses as a food preservative.

Nisin is a small peptide produced by Lactococcus lactis ssp lactis that is currently industrially produced. This preservative is often used for growth prevention of pathogenic bacteria contaminating the food products. However, the use of nisin as a food preservative is limited by its low production during fermentation. This low production is mainly attributed to the multitude of parameters influencing the fermentation progress such as bacterial cells activity, growth medium composition (namely carbon and nitrogen sources), pH, ionic strength, temperature, and aeration. This review article focuses on the main parameters that affect nisin production by Lactococcus lactis bacteria. Moreover, nisin applications as a food preservative and the main strategies generally used are also discussed.

Anti-biofilm activity of dodecyltrimethylammonium chloride microcapsules against Salmonella enterica serovar Enteritidis and Staphylococcus aureus.

Dodecyltrimethylammonium chloride (DTAC) was trapped into maltodextrins/pectin spray dried microcapsules to improve its activity against Salmonella enteritidis and Staphylococcus aureus biofilms. Two different microcapsules were prepared: uncomplexed DTAC-microcapsules (UDM), containing DTAC and maltodextrins; and complexed DTAC-microcapsules (CDM) containing DTAC complexed with pectin and maltodextrins. The minimum inhibitory concentrations (MIC) of both free and microencapsulated DTAC were investigated against S. Enteritidis and S. aureus. The MICs of DTAC were significantly lower when encapsulated. CDM treatment resulted in a 2 and 3.2 log reduction in S. aureus and S. Enteritidis biofilm culturable biomass, respectively. Microencapsulation reduced the cytotoxicity of DTAC by up to 32-fold. Free DTAC and CDM targeted the cell membrane resulting in the leakage of the intracellular molecules and subsequent cell death. The development of DTAC microcapsules reduced the amount of DTAC required to maintain the high standards of cleanliness and hygiene required in the food processing industries.

Nisin as a Food Preservative: Part 2: Antimicrobial Polymer Materials Containing Nisin.

Nisin is the only bacteriocin approved as a food preservative because of its antibacterial effectiveness and its negligible toxicity for humans. Typical problems encountered when nisin is directly added to foods are mainly fat adsorption leading to activity loss, heterogeneous distribution in the food matrix, inactivation by proteolytic enzymes, and emergence of resistance in normally sensitive bacteria strains. To overcome these problems, nisin can be immobilized in solid matrices that must act as diffusional barriers and allow controlling its release rate. This strategy allows maintaining a just sufficient nisin concentration at the food surface. The design of such antimicrobial materials must consider both bacterial growth kinetics but also nisin release kinetics. In this review, nisin incorporation in polymer-based materials will be discussed and special emphasis will be on the applications and properties of antimicrobial food packaging containing this bacteriocin.

Nisin as a Food Preservative: Part 1: Physicochemical Properties, Antimicrobial Activity, and Main Uses.

Nisin is a natural preservative for many food products. This bacteriocin is mainly used in dairy and meat products. Nisin inhibits pathogenic food borne bacteria such as Listeria monocytogenes and many other Gram-positive food spoilage microorganisms. Nisin can be used alone or in combination with other preservatives or also with several physical treatments. This paper reviews physicochemical and biological properties of nisin, the main factors affecting its antimicrobial effectiveness, and its food applications as an additive directly incorporated into food matrices.

Effect of a Vietnamese Cinnamomum cassia essential oil and its major component trans-cinnamaldehyde on the cell viability, membrane integrity, membrane fluidity, and proton motive force of Listeria innocua.

The antibacterial mechanism of a Cinnamomum cassia essential oil from Vietnam and of its main component (trans-cinnamaldehyde, 90% (m/m) of C. cassia essential oil) against a Listeria innocua strain was investigated to estimate their potential for food preservation. In the presence of C. cassia essential oil or trans-cinnamaldehyde at their minimal bactericidal concentration (2700 µg·mL(-1)), L. innocua cells fluoresced green after staining with Syto9® and propidium iodide, as observed by epifluorescence microscopy, suggesting that the perturbation of membrane did not cause large pore formation and cell lysis but may have introduced the presence of viable but nonculturable bacteria. Moreover, the fluidity, potential, and intracellular pH of the cytoplasmic membrane were perturbed in the presence of the essential oil or trans-cinnamaldehyde. However, these membrane perturbations were less severe in the presence of trans-cinnamaldehyde than in the presence of multicomponent C. cassia essential oil. This indicates that in addition to trans-cinnamaldehyde, other minor C. cassia essential oil components play a major role in its antibacterial activity against L. innocua cells.

Effect of emulsification and spray-drying microencapsulation on the antilisterial activity of transcinnamaldehyde.

Spray-dried redispersible transcinnamaldehyde (TC)-in-water emulsions were prepared in order to preserve its antibacterial activity; 5% (w/w) TC emulsions were first obtained with a rotor-stator homogeniser in the presence of either soybean lecithin or sodium caseinate as emulsifiers. These emulsions were mixed with a 30% (w/w) maltodextrin solution before feeding a spray-dryer. The antibacterial activity of TC alone, TC emulsions with and without maltodextrin before and after spray-drying were assayed by monitoring the growth at 30 °C of Listeria innocua in their presence and in their absence (control). Whatever the emulsifier used, antilisterial activity of TC was increased following its emulsification. However, reconstituted spray-dried emulsions stabilised by sodium caseinate had a higher antibacterial activity suggesting that they better resisted to spray-drying. This was consistent with observation that microencapsulation efficiencies were 27.6% and 78.7% for emulsions stabilised by lecithin and sodium caseinate, respectively.

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.

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.

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.

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.

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.

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.

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.

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.

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.