Carvacrol nanocapsules as a new antifungal strategy: Characterization and evaluation against fungi important for grape quality and to control the synthesis of ochratoxins.

Fungi are a problem for viticulture as they can lead to deterioration of grapes and mycotoxins production. Despite the widespread use of synthetic fungicides to control fungi, their impact on the agricultural ecosystem and human health demand safer and eco-friendly alternatives. This study aimed to produce, characterize and assess the antifungal activity of carvacrol loaded in nanocapsules of Eudragit® and chia mucilage as strategy for controlling Botrytis cinerea, Aspergillus flavus, Aspergillus carbonarius, and Aspergillus niger. Eudragit® and chia mucilage were suitable wall materials, as both favored the encapsulation of carvacrol into nanometric diameter particles. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested a successful incorporation of carvacrol into both nanocapsules, which was confirmed by presenting a good encapsulation efficiency and loading capacity. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses revealed adequate thermal resistance. All fungi were sensible to carvacrol treatments and B. cinerea was the most sensitive compared to the Aspergillus species. Lower concentrations of encapsulated carvacrol than the unencapsulated form were required to inhibit fungi in the in vitro and grape assays. Additionally, lower levels of carvacrol (unencapsulated or encapsulated) were used to inhibit fungal growth and ochratoxin synthesis on undamaged grapes in comparison to those superficially damaged, highlighting the importance of management practices designed to preserve berry integrity during cultivation, storage or commercialization. When sublethal doses of carvacrol were used, the growth of A. niger and A. carbonarius was suppressed by at least 45 %, and ochratoxins were not found. The nanoencapsulation of carvacrol using Eudragit® and chia mucilage has proven to be an alternative to mitigate the problems with fungi and mycotoxins faced by the grape and wine sector.

Modelling the growth of Staphylococcus aureus on cooked broccoli under isothermal conditions.

In this study, we developed predictive models describing the growth of Staphylococcus aureus on cooked broccoli florets. A pool of 3.5 log CFU/g of five S. aureus strains were inoculated on 10 g broccoli portions. The samples were then stored at 10, 20, 30 and 37 °C, and colonies were enumerated at different time intervals. Baranyi and Roberts model was fitted to the data using a Bayesian Adaptive Markov Chain Monte Carlo for estimation of the growth parameters. S. aureus showed low growth at 10 °C on broccoli samples and at 20-37 °C interval, Baranyi and Roberts model fitted well to the experimental data (R2>0.97). Estimated growth parameters were correlated with the possibility of toxin production and indicate the potential presence of these biological hazards on contaminated broccoli after heat treatment. Additionally, linear regression was performed for growth rate as storage temperature function. This secondary model followed a linear tendency with R2=0.997 and was compared with two tertiary models (ComBase Predictor and Pathogen Modeling Program) and literature data, demonstrating similar growth rate values of both. These results can be helpful for food services and managers to establish food safety standards for S. aureus growth on cooked broccoli.

Carvacrol encapsulation into nanostructures: Characterization and antimicrobial activity against foodborne pathogens adhered to stainless steel.

Carvacrol is a natural antimicrobial capable of inhibiting several microorganisms. The encapsulation of this compound may increase its stability, water solubility and provide controlled release. In this study, carvacrol encapsulated into nanoliposomes (NLC) and polymeric Eudragit® nanocapsules (NCC) was tested against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella spp. adhered to stainless steel. NLC showed an average diameter of 270.8 nm, zeta potential of +8.64 mV, and encapsulation efficiency of 98%. Minimum Bactericidal Concentration (MBC) of NLC was 3.53 mg/mL against Salmonella and 5.30 mg/mL against the other bacteria. NCC presented an average diameter of 159.3 nm, zeta potential of +44.8 mV, and encapsulation efficiency of 97%. MBC of NCC was 4.42 mg/mL against E. coli and 3.31 mg/mL against the other bacteria. After 2 h incubation with NCC at carvacrol concentration equivalent to ½ MBC, viable counts of Salmonella and E. coli were below the detection limit (1.69 CFU/mL). The population of L. monocytogenes and S. aureus was reduced by 2 log CFU/mL in 6 h. Afterwards, pools of each bacterium were separately adhered to stainless steel coupons (initial population 6.5 CFU/cm2). Salmonella and E. coli were inhibited below the detection limit using the NCC at concentration equivalent to MBC, while L. monocytogenes and S. aureus were reduced by 4 log CFU/cm2 and 3.5 log CFU/cm2, respectively. Although free carvacrol presented better results than encapsulated one in all tests performed, using encapsulated carvacrol could be more interesting for food applications by masking the strong aroma of the compound, in addition to a controlled release of carvacrol. The results suggest that NCC have potential for use in food contact surfaces in order to avoid bacterial adhesion and subsequent biofilm formation.

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.

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin.

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13 days at 4 °C, and after this period, the encapsulated pediocin retained 50 % its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190 nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80 %. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.

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.

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.

Bacteriological contamination of groundwater of the western area of Santa Catarina, Brazil / Contaminação bacteriológica de águas subterrâneas da região oeste de Santa Catarina, Brasil

The aim of this study was to evaluate the bacteriological quality of groundwater in the west region of the State of Santa Catarina, where it is common the use of private or community wells. The samples were assessed by Multiple Tube Fermentation technique according to methodology described in the American Public Health Association guidelines, and in accordance with Federal Decree 62, 26 August 2003 of Brazilian Ministry of Agriculture and Supply. A data survey was conducted by means a questionnaire which was asked to be answered by persons responsible for the wells. According to the Decree 518/2004 of the Brazilian Ministry of Health, of 212 analyzed water samples, 161 (75.94%) unsuitable for human consumption, as high rates of bacterial contamination were detected. Despite high levels of bacterial contamination in samples, the majority of consumers consider this water of good quality and, therefore only 10% of the consumers used to filter the water and 8% had the habit in boiling it before consumption. Urgent preventive actions should be provided in order to improve the quality of these well-waters, and also to set the adequate treatment of water in contaminated wells.

O objetivo do estudo foi avaliar a qualidade bacteriológica de águas subterrâneas da região oeste do Estado de Santa Catarina, onde é comum a utilização de poços comunitários ou particulares. As amostras foram avaliadas pela técnica de fermentação em tubos múltiplos de acordo com a metodologia descrita no American Public Health Association e segundo a instrução normativa 62 de 26 de agosto de 2003, do Ministério da Agricultura Pecuária e Abastecimento. Além disso, foi realizado levantamento de dados por meio de um questionário, cujas questões foram respondidas pelo responsável pelo respectivo poço. Das 212 amostras de água analisadas, 161 (75,94%) estavam impróprias para o consumo humano, conforme a Portaria 518/2004 do Ministério da Saúde. Apesar dos altos índices de contaminação bacteriológica nas amostras avaliadas, a maioria dos consumidores julga essa água de boa qualidade e por isso, apenas 10% dos usuários costumam filtrar e 8% fervem a água antes do consumo. Portanto, torna-se necessário a adoção de medidas preventivas e o tratamento das águas já comprometidas.