Effect of nanovesicle-encapsulated nisin on growth of Listeria monocytogenes in milk

Commercial nisin was encapsulated in nanovesicles (mean diameter 140 nm) prepared from partially purified soy lecithin. Nisin-loaded liposomes and unencapsulated (free) nisin were initially tested in BHI medium and skim milk inoculated with Listeria monocytogenes and incubated for 48 h at 30 °C. At such abuse temperature conditions, free nisin showed better inhibitory than the liposomal counterparts. Subsequently, the effect of encapsulated or free nisin was evaluated in combination with refrigeration (7 ± 1 °C) in both whole (3.25% fat) and skim (0% fat) milk for up to 14 day. A decrease of 3–4 log cycles in L. monocytogenes counts was observed for free and encapsulated nisin at 0.5 mg/ml concentration. Liposome encapsulation of antimicrobial peptides may be important to overcome stability issues and interaction with food components. The utilization of nanovesicle-encapsulated nisin in combination with low temperatures appeared to be effective to control L. monocytogenes in milk, emphasizing the importance of hurdle technology to assure food safety.     

Phospholipid nanovesicles containing a bacteriocin-like substance for control of Listeria monocytogenes

Bacillus licheniformis strain P40 produces a bacteriocin-like substance (BLS) that has potential to be used as a natural biopreservative for control of pathogenic and food microorganisms. The objective of this study was the encapsulation of BLS in phosphatidylcholine vesicles, evaluating its antimicrobial activity against Listeria monocytogenes. The size of the nanovesicles with the BLS was around 570 nm and of the nanovesicles without BLS was of approximately 484.8 nm, as determined by light scattering with a He–Ne laser (λ = 632.8 nm) as light source. The encapsulated BLS showed inhibitory activity against L. monocytogenes as observed by agar diffusion assay. Complete inhibition of L. monocytogenes growth was observed with the addition of 100 and 50 AU mL^− 1 of encapsulated and free BLS, respectively. A reduction in the number of viable cells to zero was observed after 10 min incubation with 400 AU mL^− 1 of either encapsulated or free BLS. The encapsulated BLS was stable for up to 30 days at 4 °C. These results indicate that nanovesicles containing BLS may have potential for use as food preservative.Industrial relevanceThe increased concern on minimally processed food and natural additives has been stimulated many studies on the utilization of antimicrobial peptides as biopreservatives. The incorporation of bacteriocins into nanovesicles may represents an interesting alternative for controlled release and increased stability of bacteriocins.     

Anti-Listeria monocytogenes effect of bacteriocin-incorporated agar edible coatings applied on cheese

Bacteriocins synthesised by lactic acid bacteria are natural alternatives to control Listeria monocytogenes, a foodborne pathogen. Additionally, antimicrobial biopolymer coatings have significant benefits in food packaging and preservation. The effect of anti-L. monocytogenes enterocins synthesised by Enterococcus avium DSMZ17511 was evaluated when supported on agar edible films and applied as antimicrobial coatings on different cheese matrices artificially contaminated with L. monocytogenes 01/155. A decrease of at least 1 log unit in viability of the pathogen was detected. The wetter matrix of soft cheese facilitated rapid diffusion of the antimicrobials, while the drier matrix of semi hard cheese produced a gradual release with prolonged inhibition of the pathogen. Also, DSMZ17511 antimicrobial peptides, only at very high concentrations, exhibited mild cytotoxicity against L929 and Caco-2 cell lines. Therefore, application of these enterocin agar coatings could be an effective, low cost, natural and safe alternative to control L. monocytogenes in cheeses.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

Microencapsulation of probiotic cells by means of rennet-gelation of milk proteins

Probiotic cells were microencapsulated in milk protein matrices by means of an enzymatic induced gelation with rennet. Water insoluble, spherical capsules with a volume-based median of 68 ± 5 μm were obtained from a novel developed emulsifying and subsequent internal gelation process. A high encapsulation yield was found due to the encapsulation procedure for Lactobacillus paracasei ssp. paracasei F19 and Bifidobacterium lactis Bb12. After incubation at low pH-values, microencapsulation yielded higher survival rates compared to non-encapsulated probiotic cells. The viable cell numbers of encapsulated Lactobacillus paracasei and Bifidobacterium lactis were 0.8 and 2.8 log units CFU g⁻¹ higher compared to free cells after 90 min incubation at pH 2.5. The improved survival of encapsulated cells can probably be explained by a higher local pH-value within the protein matrix of the capsules caused by the protein buffering capacity, protecting the cells during incubation under simulated gastric conditions at low pH. The study indicates that rennet-induced gelation of skim-milk concentrates for the microencapsulation of probiotic cells can be a suitable alternative to current available technologies, mainly based on ionotrophic gelation of plant-polymer solutions.     

Encapsulation of probiotic Lactobacillus acidophilus by ionic gelation with electrostatic extrusion for enhancement of survival under simulated gastric conditions and during refrigerated storage

The probiotic Lactobacillus acidophilus was encapsulated in biodegradable and biocompatible capsules prepared by ionic gelation between phytic acid (PA) and chitosan (CS) with an electrostatic extrusion method. Calcium carbonate (CaCO₃) and starch were used as co‐encapsulants for improvement of capsule stability. Capsules were characterised and evaluated for survival of encapsulated L. acidophilus cells in simulated gastric fluid (SGF) and during refrigerated storage. Loading capacity values of PA‐CS capsules, PA‐CS‐starch capsules and PA‐CS‐CaCO₃ capsules were 8.20, 8.12 and 7.81 log CFU g^?1 of wet capsule, respectively. Capsules showed particle sizes of 1.3–1.5 mm and a uniform spherical shape. PA‐CS‐CaCO₃ capsules were the most stable vehicles for the protection of probiotic cells against acidic damage, particularly at pH 1.5 and pH 2. L. acidophilus cells from PA‐CS‐CaCO₃ capsules showed only a 0.64 log CFU reduction in numbers after 2 h in pH 1.5 SGF conditions. The numbers of L. acidophilus encapsulated in PA‐CS‐CaCO₃ capsules were decreased by only 0.69 log CFU g^?1, while PA‐CS capsules and PA‐CS‐starch capsule numbers were reduced by more than 1.45 log CFU g^?1 after 4 weeks at 4 °C. Addition of calcium carbonate to PA‐CS capsules provided protection against acid injury via antacid and buffering effects for encapsulation of L. acidophilus.     

Control of Growth of L. monocytogenes in Fresh Salmon using Microgard and Nisin

Pathogens like Listeria monocytogenes in fresh, or even in cold smoked salmon, have become a major public health concern for the salmon processing industry and government agencies. The effect of bacteriocin solutions (Microgard^™ and Nisin) on reducing total microbial counts, inhibiting Listeria monocytogenes, and prolonging the shelf-life was evaluated. Listeria monocytogenes was inoculated onto chilled and on frozen and thawed salmon samples. The combination of Nisin and Microgard reduced the total aerobic bacteria populations of fresh chilled salmon by 2 log (P<0.05) and increased its shelf-life, at 6 °C, by 3-4 d, as compared with the control. The above bacteriocin combination also reduced the growth of inoculated Listeria monocytogenes in frozen-thawed salmon and increased its shelf-life from 5 to 10 d at 6 °C. The bacteriocin treatment did not affect surface pH values or color of the fish.     

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 °C and 37 °C, reaching a maximum production of 1.0 × 10⁹ AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.     

Development and characterization of phosphatidylcholine nanovesicles containing the antimicrobial peptide nisin

Two methodologies were compared to encapsulate nisin in liposomes of partially purified soybean phosphatidylcholine: reversed-phase and hydration film. In the hydration film method, both probe-type and bath-type ultrasound were evaluated. The size of liposomes was evaluated by light scattering analysis and residual antimicrobial activities by agar diffusion assay using Listeria monocytogenes ATCC 7644 as indicator strain. The size of liposomes prepared by reversed-phase, hydration film using probe-type and bath-type ultrasound were 190, 181 and 148 nm with residual antimicrobial activities after encapsulation of 25%, 50% and 100%, respectively. The methodology of film hydration using bath-type ultrasound was chosen for assessment of its physicochemical characteristics. Nisin had entrapment efficiency of 94.12%. Measured Zeta potentials for unfiltered and filtered (0.22 μm) liposomes were ?55.8 and ?54.5 mV, respectively. The antimicrobial activity of free nisin, encapsulated nisin and filtered was evaluated for a period of 24 days. It was observed that the free nisin remained 100% of residual activity while the liposomes containing nisin were losing their antimicrobial activity over time reaching 25% residual activity after 10 days. The size (132–149 nm) and pH (4.5) remained constant over time. It was observed by microscopy that the liposomes maintained their spherical morphology. The stability observed by size and pH was not the same regarding antimicrobial activity and Zeta potential, indicating that the liposomes should be applied shortly after its preparation.     

Characterization of semipurified enterocins produced by Enterococcus faecium strains isolated from raw camel milk

Food safety has become an issue of great interest worldwide. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to control in the dairy industry. The use of lactic acid bacteria (LAB) and their antimicrobial substances against Listeria is promising in food applications. Here, we report the isolation from raw camel milk of LAB displaying antilisterial activity. Two isolates were selected for their secretion of bacteriocin(s) and identified by 16S rRNA sequencing as Enterococcus faecium S6 and R9. The produced bacteriocins were partially purified by ammonium sulfate precipitation and then biochemically characterized. Antimicrobial activity was estimated to be 6,400 and 400 AU (arbitrary units)/mL for E. faecium S6 and R9, respectively. The proteinaceous nature of the bacteriocins was confirmed via enzymatic reactions. Moreover, lipolytic and glycolytic enzymes completely inactivated the antimicrobial effect of the bacteriocins. These bacteriocins were heat-resistant and stable over a wide range of pH (2.0 to 10.0). To confirm its inactivation by lipolytic and glycolytic enzymes, the bacteriocin of E. faecium S6 was further purified by gel filtration, which suggested the existence of carbohydrate and lipid moieties. In addition, enterocin-coding genes were identified by PCR, showing DNA fragments corresponding in size to enterocins A, B, and P for E. faecium S6 and to enterocins B and P for E. faecium R9. In conclusion, these results indicate that partially purified bacteriocins from E. faecium S6 and R9 may be beneficial in controlling Listeria in the dairy industry.     

Nanoencapsulation of Zataria multiflora essential oil preparation and characterization with enhanced antifungal activity for controlling Botrytis cinerea,the causal agent of gray mould disease

This study was undertaken to investigate the nanoencapsulation of Zataria multiflora essential oil (ZEO) in chitosan nanoparticles (CSNPs) in order to enhance antifungal activity and stability of the oils against one isolate of Botrytis cinerea Pers., the causal agent of gray mould disease. ZEO was encapsulated by an ionic gelation technique into CSNPs with an average size of 125–175 nm as observed by transmission electron microscopy (TEM). From UV-vis spectrophotometry results, the drug encapsulation and loading efficiency of ZEO decreased from 45.24% to 3.26% and from 9.05% to 5.22%, respectively, upon increasing initial ZEO content from 0.25 to 1 g/g chitosan. In vitro release studies also demonstrated a controlled and sustained release of ZEO for 40 days. The superior performance of ZEO when encapsulated by CSNPs under both in vitro and in vivo conditions in comparison with unmodified ZEO against B. cinerea was revealed. The in vivo experiment also showed that the encapsulated oils at 1500 ppm concentration significantly decreased both disease severity and incidence of Botrytis-inoculated strawberries during 7 days of storage at 4 °C followed by 2–3 more days at 20 °C. These findings revealed the promising role of CSNPs as a controlled release system for EOs in order to enhance antifungal activities.Industrial relevance: Application of plant essential oil (EOs) treatment at pre- or postharvest stage has been considered as an alternative treatment to the use of synthetic fungicides to prevent fruit postharvest decay and to extend the storage life while retaining the overall quality of different fresh commodities. Although EOs have proved to be good antimicrobial agents, their use for maintaining fruit quality and reducing fungal decay is often limited due to their volatile compounds which can easily suffer degradation under the action of heat, pressure, light and oxygen. Furthermore, they are insoluble in water, and for certain applications a controlled release is required. In this regard, nano-size carriers provide more surface area and can possibly upgrade solubility, enhance bioavailability and improve controlled release and targeting of the encapsulated food ingredients, in comparison to micro-size carriers. These findings revealed the promising role of CSNPs as a controlled release system for EOs in order to enhance their antimicrobial activities.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.     

Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81

Enterococcus faecium WHE 81, isolated from cheese, has been reported to produce a bacteriocin called “enterocin 81” [J. Appl. Microbiol. 85 (1998) 521.]. Purification of “enterocin 81” was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C₂/C₁₈ reverse-phase chromatographies. The antimicrobial was eluted from the C₂/C₁₈ column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4833.0 and 5462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively. Using proper primers, chromosomal fragments of 212 and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.     

Transglutaminase-induced caseinate gelation for the microencapsulation of probiotic cells

A novel method for the encapsulation of probiotic cells in foodgrade casein microcapsules was developed. The process is based on a transglutaminase-catalysed gelation of casein suspensions containing probiotic cells. Water insoluble, spherical capsules with a volume-based median diameter of 165 ± 23 μm resulted from the process. Encapsulation yields of 70 ± 15% and 93 ± 22% were achieved for Lactobacillus paracasei ssp. paracasei F19 and Bifidobacterium lactis Bb12, respectively. Analysis of living cell numbers after incubation of free and encapsulated probiotics in simulated gastric juice without pepsin at pH 2.5 and pH 3.6 (37 °C, 90 min) showed a protective effect due to microencapsulation under all conditions tested. The study indicates that transglutaminase-induced caseinate gelation can be applied to the microencapsulation of probiotics. Furthermore, it could be shown that an entrapment in a dense casein matrix can protect these microorganisms from damage due to pH-levels similar to those in the human stomach.     

Antimicrobial activity against foodborne pathogens of chitosan biopolymer films of different molecular weights

Antimicrobial activity against Listeria monocytogenes, Escherichia coli 0157:H7 and Samonella typhimurium of chitosan biopolymer films (CBFs) prepared with four different viscosities of chitosans (10, 40, 100 and 200 mPa s) were investigated by agar diffusion assay. The films were also characterized with measurements of color, tensile strength (TS), % elongation (EL), water vapor permeability and oxygen permeability. CBFs prepared with 100 mPa s chitosan showed an antimicrobial effect only on 10⁴ cfu/mL inoculation of L. monocytogenes while other viscosities showed an antilisterial effect on all concentrations (10⁴-10⁶ cfu/mL) of L. monocytogenes. CBFs prepared with 10 mPa s (CBF-10) and 40 mPa s (CBF-40) chitosans showed an inhibitory effect against E. coli 0157:H7 and S. typhimurium only at the 10⁴ cfu/mL concentration. CBFs prepared with the two higher viscosity chitosans did not show any effect regardless of bacterial level. TS and EL of the CBFs increased with increasing viscosity up to 100 mPa s. Molecular weight distribution was found to be positively correlated with viscosity. The oxygen permeability of the CBFs increased with increasing viscosity of chitosans, but water vapor transmission rate was not similarly affected. In conclusion, CBFs were more effective at inhibition of L. monocytogenes than S. typhimurium and E. Coli O157:H7. Molecular weight of chitosan must be chosen selectively to control the target foodborne pathogens.     

Efficacy of freezing, frozen storage and edible antimicrobial coatings used in combination for control of Listeria monocytogenes on roasted turkey stored at chiller temperatures

The presence and growth of Listeria monocytogenes on ready-to-eat (RTE) turkey is an important food safety issue. The antilisterial efficacy of four polysaccharide-based edible coatings (starch, chitosan, alginate and pectin) incorporating sodium lactate (SL) and sodium diacetate (SD) as well as commercial preparations Opti.Form PD4, NovaGARD™ CB1, Protect-M and Guardian™ NR100 were compared against L. monocytogenes on roasted turkey. Pectin coating treatments incorporating SL/SD, Opti.Form PD4 with or without Protect-M, and NovaGARD™ CB1 displayed higher antimicrobial efficacy against.L. monocytogenes than the other antimicrobials and coating materials. In the second phase of the study, it was investigated whether frozen storage could enhance the antilisterial effectiveness of pectin coating treatments on chilled roasted turkey. Inoculated roasted turkey samples coated with pectin-based treatments were frozen for up to 4 weeks and subsequently stored at 4 °C for 8 weeks. Frozen storage significantly enhanced the antilisterial activity of various coating treatments; with selected treatments reducing the L. monocytogenes populations by as much as 1.1 log CFU/cm² during the subsequent 8-week chilled storage. This study demonstrates that pectin-based antimicrobial edible coatings hold promise in enhancing the safety of RTE poultry products and frozen storage has the potential to enhance their effectiveness.     

Liposomal incorporation of carvacrol and thymol isolated from the essential oil of Origanum dictamnus L. and in vitro antimicrobial activity

The chemical composition of the essential oils from Origanum dictamnus L. (wild and organic cultivated plant) was analysed by GC–MS. Carvacrol, thymol, p-cymene, and γ-terpinene were identified as major constituents and isolated from both samples. The above components were successfully encapsulated in phosphatidyl choline-based liposomes and the possible improvement of their antioxidant and antimicrobial activities was tested against selected microbia. The antimicrobial properties of the oils were tested by a diffusion technique against four Gram positive and four Gram negative bacteria and three human pathogenic fungi, as well as the food-borne pathogen, Listeria monocytogenes. The percentage of the encapsulated carvacrol, the major component of the oil, was determined by GC–FID. In order to investigate any possible synergistic or antagonistic effect between carvacrol/thymol and carvacrol/γ-terpinene, the antimicrobial activities of the mixtures, were also determined before and after encapsulation in liposomes. All tested compounds presented enhanced antimicrobial activities after the encapsulation. Finally, in all cases, their antioxidant activity using differential scanning calorimetry was studied, in order to gain knowledge about their oxidation stability.     

Application of Central Composite Design to evaluate the antilisterial activity of hydro-alcohol berry extract of Myrtus communis L.

The antimicrobial activity of the hydro-alcohol extract of Myrtus communis L. (ME) berries was investigated against six Listeria monocytogenes strains (2 type strains and 4 isolates). Sub-lethal ME concentrations reduced L. monocytogenes counts by at least 2 log cycles. A Central Composite Design was used to investigate the combined effects of sub-lethal concentrations of ME (0.039–0.195 mL/100 mL), NaCl (0–2.0 g/100 mL) and pH (5.0–7.0) on strains growth. ME affected growth parameters, generally extending lag phase length and reducing maximum growth, sometimes with interactive effects with pH. The highest ME concentrations (0.117–0.195 mL/100 mL) combined with the lowest pH values (5.0–6.0) strongly reduced or even inhibited strains growth.     

Application of bacteriocin-producing Enterococcus faecium isolated from donkey milk,in the bio-control of Listeria monocytogenes in fresh whey cheese

A total of 79 bacterial strains, previously isolated from donkey milk, were screened for their antimicrobial activity against several spoilage and foodborne pathogenic bacteria. Amongst them, 3 strains belonging to Enterococcus faecium displayed antimicrobial activity against Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. Mass spectrometry analysis demonstrated that all enterococci used in this study produced peptides with masses consistent with those for enterocins A and B. The cell-free supernatants of the identified bacteriocin-producing enterococci were equally active over a wide range of pH and heat treatments making them excellent candidates for potential applications in bio-preservation. Bacteriocins produced by these strains were tested for their capability to control post-processing contamination and growth of L. monocytogenes during refrigerated storage of artificially contaminated fresh whey cheese. One strain was considered bactericidal while the other two were classified as bacteriostatic.