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.

Cold plasma surface treatments to prevent biofilm formation in food industries and medical sectors.

Environmental conditions in food and medical fields enable the bacteria to attach and grow on surfaces leading to resistant bacterial biofilm formation. Indeed, the first step in biofilm formation is the bacterial irreversible adhesion. Controlling and inhibiting this adhesion is a passive approach to fight against biofilm development. This strategy is an interesting path in the inhibition of biofilm formation since it targets the first step of biofilm development. Those pathogenic structures are responsible for several foodborne diseases and nosocomial infections. Therefore, to face this public health threat, researchers employed cold plasma technologies in coating development. In this review, the different factors influencing the bacterial adhesion to a substrate are outlined. The goal is to present the passive coating strategies aiming to prevent biofilm formation via cold plasma treatments, highlighting antiadhesive elaborated surfaces. General aspects of surface treatment, including physico-chemical modification and application of cold plasma technologies, were also presented. KEY POINTS: • Factors surrounding pathogenic bacteria influence biofilm development. • Controlling bacterial adhesion prevents biofilm formation. • Materials can be coated via cold plasma to inhibit bacterial adhesion.

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.

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.

Cold plasma assisted deposition of organosilicon coatings on stainless steel for prevention of adhesion of Salmonella enterica serovar Enteritidis.

The persistence of Salmonella enterica on abiotic surfaces in hospitals and the agri-food industries leads to several infections worldwide. In this context, this work aimed to study the adhesion of S. Enteritidis on plasma-modified stainless steel to prevent biofilm-associated-infections. Surface modification was achieved by the elaboration of organosilicon coatings from the monomer 1,1,3,3-tetramethyldisiloxane, mixed with oxygen, using a flowing nitrogen microwave post-discharge plasma polymerization technique. The effect of cold plasma parameters on the properties of the coatings, the coated surface topography and S. Enteritidis cell adhesion was studied. The results showed that the surface topography influenced the bacterial adhesion rate. Indeed, rough surfaces did not repel S. Enteritidis since the number of attached cells on these coatings was between 30 ± 4 to 65 ± 4 bacteria per microscopic field. Otherwise, smoother surfaces demonstrated an anti-adhesive character since the number of attached cells was almost nil on these coatings.

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.

Actively detached Pseudomonas aeruginosa biofilm cell susceptibility to benzalkonium chloride and associated resistance mechanism.

The present work aimed at studying physiological properties of Pseudomonas aeruginosa cells actively detached from biofilm formed on stainless steel and comparing them with their planktonic counterparts as a function of growth temperature (20 °C and 37 °C). The susceptibility of P. aeruginosa cells to benzalkonium chloride (BAC) was studied. Furthermore, the effect of BAC on the cell membrane integrity and the role of the cell membrane fluidity in the cell-scale-resistance mechanism were investigated. Our results showed that actively detached biofilm cells were more susceptible to BAC treatment than planktonic ones. A greater leakage of intracellular potassium after BAC addition was observed in actively detached biofilm cells, which reflects their membrane vulnerability. The rise of the growth temperature from 20 to 37 °C increased the membrane rigidity of planktonic cells comparatively to their actively detached biofilm ones. Under experimental conditions developed in this work, our data highlighted that actively biofilm-detached and planktonic P. aeruginosa cells have distinguishable phenotypes.

Impact of growth temperature on the adhesion of colistin-resistant Escherichia coli strains isolated from pigs to food-contact-surfaces.

This study investigated the effect of the growth temperature (20 and 37 °C) of Escherichia coli strains isolated from pigs on their adhesion to stainless steel and polycarbonate. This study also evaluated the ability of the DLVO and XDLVO mathematical models to predict this adhesion. The rise of growth temperature from 20 to 37 °C significantly influenced the adhesion of studied E. coli strains. The data also underlined that the mathematical prediction did not fully match with the experimental bacterial adhesion to surfaces. Furthermore, results showed that the colistin-resistant and sensitive E. coli strains adhesion depends on the type of abiotic surface. Based on these results, the mathematical models are limited in the prediction of the bacterial adhesion to abiotic surfaces. The surface roughness is a major parameter of the bacterial adhesion and should be included in the future mathematical models predicting the bacterial adhesion.

Vaginal Lactobacillus gasseri CMUL57 can inhibit herpes simplex type 2 but not Coxsackievirus B4E2.

This study aimed at demonstrating the antiviral activity of Lactobacillus gasseri CMUL57 (L. gasseri CMUL57), L. acidophilus CMUL67 and L. plantarum CMUL140 against herpes simplex type 2 (HSV-2) and Coxsackievirus B4E2 (CVB4E2), which are enveloped and naked viruses, respectively. These lactobacilli were non-cytotoxic and were able to reduce the cytopathic effect induced by HSV-2 in Vero cell monolayers. However, lactobacilli were not active against CVB4E2. Tested lactobacilli displayed anti-HSV-2 activity when they were co-incubated with the virus prior to inoculating the mixture to Vero cell monolayers. The detection of HSV-2 DNA by PCR in pellets of bacteria/virus mixtures let us to hypothesize that anti-HSV-2 activity of lactobacilli resulted from the viruses' entrapment. This study showed the capabilities of vaginal lactobacilli to inhibit enveloped viruses such as HSV-2.

Effect of culture conditions on the resistance of Pseudomonas aeruginosa biofilms to disinfecting agents.

The relationship between the environmental conditions of biofilm formation and resistance to disinfectants was studied. Anti-biofilm assays were performed against biofilms grown at 20, 30 and 37°C on stainless steel and polycarbonate, over 24 and 48 h. A rise in growth temperature increased the resistance of 24 h biofilms to disinfectants containing didecyldimethylammonium chloride and decreased it to a disinfectant containing alkyldimethylbenzylammonium chloride. The increase in growth temperature coupled with an incubation time of 24 h promoted increases in both matrix production and the membrane rigidity of sessile cells. An increase in incubation time also increased both matrix production and the membrane rigidity of sessile cells. Such phenomena resulted in an increased resistance to disinfectants of biofilms grown at 20 and 30°C. The resistance of 48 h biofilms to disinfectants decreased with an increase in growth temperature despite the increase in matrix production and the membrane rigidity of sessile cells.

Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments.

The biofilm formation on abiotic surfaces in food and medical sectors constitutes a great public health concerns. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodborne and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food and medical area, seem also to enhance the biofilm formation and their resistance to disinfectant agents. In this regard, this review highlights the effect of environmental conditions on bacterial adhesion and biofilm formation on abiotic surfaces in the context of food and medical environment. It also describes the current and emergent strategies used to study the biofilm formation and its eradication. The mechanisms of biofilm resistance to commercialized disinfectants are also discussed, since this phenomenon remains unclear to date.

Identification of vaginal lactobacilli with potential probiotic properties isolated from women in North Lebanon.

The aim of this work was to study the diversity of vaginal lactobacilli in Lebanese women and to evaluate the antagonism, hydrophobicity, and safety characteristics of these strains. This study was performed on samples from 135 women who visited a gynecology clinic in the north of Lebanon, between September 2012 and January 2013. From these samples, 53 different isolates of vaginal lactobacilli were collected from vaginal swabs and identified using biochemical and molecular methods. The use of genotypic Rep-PCR fingerprinting allowed for the organization of these isolates into 23 different groups. Seven of the isolated lactobacilli were antagonistic against the following vaginal pathogens: Gardnerella vaginalis CIP7074T, Staphylococcus aureus ATCC33862, Escherichia coli CIP103982, and Candida albicans ATCC10231. The antagonistic lactobacilli strains were then identified using 16S rDNA sequence. The data of this study show that the antagonistic lactobacilli were non-hemolytic, sensitive to most antibiotic tests, free of plasmid DNA, and exhibited interesting hydrophobicity and autoaggregation properties positioning them as potential candidates for probiotic design.

Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants.

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.

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.

Nisin adsorption on hydrophilic and hydrophobic surfaces: evidence of its interactions and antibacterial activity.

Study of peptides adsorption on surfaces remains a current challenge in literature. A complementary approach, combining X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to investigate the antimicrobial peptide nisin adsorption on hydrophilic and hydrophobic surfaces. The native low density polyethylene was used as hydrophobic support and it was grafted with acrylic acid to render it hydrophilic. XPS permitted to confirm nisin adsorption and to determine its amount on the surfaces. ToF-SIMS permitted to identify the adsorbed bacteriocin type and to observe its distribution and orientation behavior on both types of surfaces. Nisin was more oriented by its hydrophobic side to the hydrophobic substrate and by its hydrophilic side to the outer layers of the adsorbed peptide, in contrast to what was observed on the hydrophilic substrate. A correlation was found between XPS and ToF-SIMS results, the types of interactions on both surfaces and the observed antibacterial activity. Such interfacial studies are crucial for better understanding the peptides interactions and adsorption on surfaces and must be considered when setting up antimicrobial surfaces.

Nisin-activated hydrophobic and hydrophilic surfaces: assessment of peptide adsorption and antibacterial activity against some food pathogens.

An effective antimicrobial packaging or food contact surface should be able to kill or inhibit micro-organisms that cause food-borne illnesses. Setting up such systems, by nisin adsorption on hydrophilic and hydrophobic surfaces, is still a matter of debate. For this purpose, nisin was adsorbed on two types of low-density polyethylene: the hydrophobic native film and the hydrophilic acrylic acid-treated surface. The antibacterial activity was compared for those two films and it was highly dependent on the nature of the surface and the nisin-adsorbed amount. The hydrophilic surfaces presented higher antibacterial activity and higher amount of nisin than the hydrophobic surfaces. The effectiveness of the activated surfaces was assessed against Listeria innocua and the food pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. S. aureus was more sensitive than the three other test bacteria toward both nisin-functionalized films. Simulation tests to mimic refrigerated temperature showed that the films were effective at 20 and 4 °C with no significant difference between the two temperatures after 30 min of exposure to culture media.

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.

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.

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.