Patrícia da Silva Malheiros

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 degrees 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 degrees 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.

Effect of liposome-encapsulated nisin and bacteriocin-like substance P34 on Listeria monocytogenes growth in Minas frescal cheese

The efficacy of liposome-encapsulated nisin and bacteriocin-like substance (BLS) P34 to control growth of Listeria monocytogenes in Minas frescal cheese was investigated. Nisin and BLS P34 were encapsulated in partially purified soybean phosphatidylcholine (PC-1) and PC-1-cholesterol (7:3) liposomes. PC-1 nanovesicles were previously characterized. PC-1-cholesterol encapsulated nisin and BLS P34 presented, respectively, 218 nm and 158 nm diameters, zeta potential of -64 mV and -53 mV, and entrapment efficiency of 88.9% and 100%. All treatments reduced the population of L. monocytogenes compared to the control during 21 days of storage of Minas frescal cheese at 7°C. However, nisin and BLS P34 encapsulated in PC-1-cholesterol liposomes were less efficient in controlling L. monocytogenes growth in comparison with free and PC-1 liposome-encapsulated bacteriocins. The highest inhibitory effect was observed for nisin and BLS P34 encapsulated in PC-1 liposomes after 10 days of storage of the product. The encapsulation of bacteriocins in liposomes of partially purified soybean phosphatidylcholine may be a promising technology for the control of foodborne pathogens in cheeses.

Adhesion and biocides inactivation of Salmonella on stainless steel and polyethylene.

The adhesion of Salmonella (S.) strains to stainless steel and polyethylene and their inactivation by biocides used in food industry was investigated. Coupons of stainless steel and polyethylene were immersed in bacterial suspensions of S. Enteritidis, S. Typhimurium, and S. Bredeney during 15, 30, and 60 minutes, and submitted to different concentrations of peracetic acid (PAA), sodium hypochlorite (NaOCl), and quaternary ammonium (Quat) sanitizers. Hydrophobicity of the surfaces was evaluated by contact angle measurements using the sessile drop method and bacterial adhesion was accompanied through bacterial counts and scanning electron microscopy (SEM). Results indicated that the three serovars of Salmonella presented similar adhesion to both materials (5.0 to 6.5 log cfu cm -2 ). The time of exposure did not influence the counts of adhered cells on both surfaces, however SEM revealed larger clusters of S. Enteritidis on both materials, not found for the other serovars. S. Enteritidis presented lower sessile drop angle on polyethylene, indicating hydrophilic properties of this material. The biocides were not able to inactivate all the microorganisms adhered on both surfaces. At least 1 log cfu cm -2 of all serovars tested remained viable after the exposure to different biocide concentrations. In general, higher counts of survivors were observed on polyethylene disinfected with different concentrations of biocides. S. Bredeney e S. Typhimurium were more resistant than S. Enteritidis to PAA, whilst S. Enteritidis presented smaller reduction rates to NaOCl. This last biocide was able to reduce Salmonella counts in approximately 3.0 to 4.0 log cm -2 . When adhered to polyethylene, the serovars S. Typhimurium and S. Enteritidis were more resistant to Quat than S. Bredeney in all concentrations tested, and the numbers of S. Enteritidis remained almost unaltered. On stainless steel disinfected by Quat, S. Bredeney presented higher numbers of survivors.

Inhibition of listeria monocytogenes in minas frescal cheese by free and nanovesicle-encapsulated nisin

The effectiveness of free and nanovesicle-encapsulated nisin to control Listeria monocytogenes in Minas Frescal cheese was investigated. Commercial nisin was encapsulated into liposomes of partially purified soy lecithin. Free (0.1 mg/mL and 0.25 mg/mL) and nanovesicle-encapsulated nisin (0.25 mg/mL) were applied onto the surface of cheese samples, and L. monocytogenes was inoculated before incubation at 6-8°C for 28 days. A bactericidal effect was observed with 0.25 mg/mL free nisin; a bacteriostatic effect was observed for liposome-encapsulated nisin and 0.1 mg/mL free nisin. Free nisin was more efficient than nisin-loaded liposomes in controlling L. monocytogenes. Possible reasons for this behavior, and also the significance of nisin to soft cheeses are discussed. Nisin acted as a suitable barrier within hurdle technology, potentially extending the shelf-life and safety of fresh cheeses.

Antimicrobial activity of free and liposome-encapsulated thymol and carvacrol against Salmonella and Staphylococcus aureus adhered to stainless steel

Antimicrobial activity of thymol, carvacrol and thymol/carvacrol liposomes (TCL) was evaluated against two bacterial pools, each one consisting of four strains of Staphylococcus aureus or Salmonella enterica. TCL were prepared using thin-film hydration, showing 270.20nm average diameter (polydispersity index of 0.33) and zeta potential of +39.99mV. Minimum inhibitory concentration (MIC) of thymol, carvacrol and TCL against S. aureus pool was 0.662mg/ml, while MIC for Salmonella pool was 0.331mg/ml for thymol and carvacrol, and for TCL was 0.662mg/ml. Bacterial pools (8.0logCFU/ml), allowed in contact on stainless steel AISI 304 coupons in UHT skim milk for 15min, resulted in adhered populations of 5.6-6.1logCFU/cm2. Adhered S. aureus (±6.1logCFU/cm2) were inhibited after 1-min and 10-min treatments using thymol or carvacrol at MIC and 2.0 MIC. Reductions of 1.47-1.76logCFU/cm2 and 1.87-2.04logCFU/cm2 were obtained using 0.5 MIC of thymol and carvacrol, respectively. A 10-min contact with free (MIC and 2.0 MIC) and encapsulated (MIC) antimicrobials inhibited attached Salmonella (±6.0logCFU/cm2); however, after 1-min of contact, 2.0 MIC of thymol and carvacrol were not able to inactivate adhered Salmonella MIC of TCL inactivated S. aureus and Salmonella after 10min; however, after 1-min contact, adhered S. aureus and Salmonella populations were decreased in 1.62logCFU/cm2 and 2.01logCFU/cm2, respectively. Considering antimicrobial concentrations and contact times, thymol, carvacrol, and TCL could be employed in food-contact surfaces to prevent biofilm formation at early stages of bacterial attachment. Further investigations should be performed considering long-term antibacterial effects of TCL.

Antagonistic activity of Lactobacillus acidophilus LA10 against Salmonella enterica serovar Enteritidis SE86 in mice

Salmonella enterica serovar Enteritidis is one of the main pathogens responsible for foodborne illness in Brazil. Probiotic bacteria can play a role in defense and recovery from enteropathogenic infections. In this study, the ability of Lactobacillus acidophilus LA10 to colonise and exert antagonistic effects in the gastrointestinal tract was tested before and during experimental infection in conventional mice contaminated with S. Enteritidis (SE86). A dose of 0.1 mL containing 108 viable cells of SE86 and L. acidophilus LA10 was orally administered by gavage to mice. The experiment was divided into groups. As a negative control, Group 1 was administered only sterile saline solution. As a positive control, Group 2 was administered only SE86. Group 3 was first administered SE86, and after 10 days, treated with L. acidophilus LA10. Group 4 was first administered L. acidophilus LA10, and after 10 days, challenged with SE86. The results demonstrated that a significant number of SE86 cells were able to colonize the gastrointestinal tract of mice, specifically in the colon and ileum. L. acidophilus LA10 demonstrated an antagonistic effect against SE86, with better results observed for Group 3 over Group 4. Thus, L. acidophilus LA10 shows potential antagonistic effects against S. Enteritidis SE86, especially if administered after infection.

Optimization of growth and bacteriocin production by Lactobacillus sakei subsp. sakei2a

Lactobacillus sakei subsp. sakei 2a is a bacteriocinogenic lactic acid bacterium isolated from Brazilian pork sausage, capable of inhibiting the growth of microbial pathogens, mainly Listeria monocytogenes. In order to optimize bacteriocin production for industrial applications, this study evaluated the effect of supplementation of MRS broth with glucose, Tween 20, Tween 80, sodium citrate, potassium chloride and cysteine, and effect of the initial pH and temperature of incubation of the medium on production of bacteriocins by L. sakei 2a. Adding glucose and Tween 20 to the medium, an initial pH of 5.0 or 5.5, and incubation temperatures of 25 °C or 30 °C resulted to the highest bacteriocin yields. Thus, a 24 factorial design with the four variables was performed, and statistical analysis showed that it was an adequate model (R 2 = 0.8296). In the studied range, the four parameters significantly influenced bacteriocin production, with the maximum yield produced at an initial pH between 5.5 and 7.0, a temperature between 25 and 30 °C and supplementation of the MRS broth with glucose from 3.25 to 6.0 g L−1 and Tween 20 from 0.575 to 1.15% (v/v). Response Surface Methodology analysis indicated that the highest bacteriocin production (12800 AU mL−1) occurred in the MRS broth supplemented with 5.5 g L−1 glucose and 1.05% Tween 20 at an initial pH of 6.28 and an incubation temperature of 25 °C. The amount of bacteriocin produced in commercial MRS broths under the same conditions was only 5600AU mL−1.