Preliminary Quantitative Microbial Risk Assessment for Staphylococcus enterotoxins in fresh Minas cheese,a popular food in Brazil

The main objective of this work was to assess the risk associated with staphylococcal enterotoxins (SE) intoxication after the consumption of fresh Minas cheese by the Brazilian population. Coagulase-positive staphylococci data from 350 samples were obtained from monitoring programs, and were used as a proxy for S. aureus contamination, considering that 73% of the strains were toxigenic. The Combined Database for Predictive Microbiology (ComBase) and the Pathogen Modeling Program (PMP) models were used to predict S. aureus growth rate and lag-phase in fresh Minas cheese at different pH, salt concentration and storage temperature in a household refrigerator, up to 7 days before consumption. Change in storage temperature had the largest impact on the growth rate and lag-phase obtained from both models. Cumulative probability of SE intake events equal to or higher than the toxigenic dose of 100 ng were calculated using Monte Carlo simulations performed by the @Risk software. The toxic dose was exceeded at the 99.95th percentile of exposure in the ComBase model (upper bound) for the adult population, the lowest percentile identified in the study. The S. aureus initial concentration was the parameter that most impacted the output obtained by @risk, indicating the importance of good manufacturing practices for fresh Minas cheese production, and proper storage conditions at the point of sale. This preliminary assessment indicated that the risk of staphylococcal intoxications from the consumption of fresh Minas cheese by the Brazilian population is probably low. The study identified many data gaps that needs to be addressed to improve the risk assessment.     

Quantitative risk assessment of Mycobacterium avium subsp. paratuberculosis survival in pasteurized milk in three dairy plants in Italy

The objective of this study was to carry out a quantitative risk assessment (QRA) of Mycobacterium avium subsp. paratuberculosis (MAP) survival in pasteurized milk produced by industrial dairy plants. Data were collected in three dairy plants (A, B and C) located in three different Italian regions and processing 38.75 (plant A), 89.29 (plant B) and 190.56 million litres (Plant C) of milk yearly. Plants A and plant C produce pasteurized milk, soft and hard industrial cheeses and yogurt; plant B produces only pasteurized milk. In-line milk filter (ILMF) samples and/or bulk milk samples were collected from all 569 herds delivering milk to the three dairy plants. Samples were analysed by quantitative real-time PCR (qPCR). The QRA considered the presence of MAP in ILMF and in bulk milk of all the dairy herds delivering milk to the three investigated dairy plants, estimating MAP concentration in raw milk on the basis of these data, the dilution effect due to mixing milk in collecting trucks and in plant silos, and the effect of pasteurization in reducing the MAP load. The expected fraction of litres of pasteurized milk with 0 MAP would be 99.02%, 99.45% and 99.12%, in plants A, B and C respectively, and an overall percentage 0.55% to 0.98% of pasteurized milk having a MAP contamination >0 colony forming units (CFU)/l and 0.04%–0.11% of pasteurized litres with a MAP contamination > 100 CFU/l was predicted. A daily variation was observed in the proportion of MAP-contaminated litres of milk. The study demonstrated that milk in the dairy plants investigated may be a source of MAP exposure for humans. The between-herd and within-herd MAP apparent prevalence in the investigated areas are likely comparable to those in other areas in Italy, Europe and North America, and the results are applicable to other geographical areas.     

Adhesion of Staphylococcus aureus on stainless steel treated with three types of milk

Staphylococcus aureus has the ability to adhere and to form biofilm on inert surface such as stainless steel commonly used in food industry. The biofilm formed on the surface of milk processing equipments could be a source of dairy products contamination. This contamination causes a food poisoning. In this paper the S. aureus adhesion on stainless steel treated by three types of milk (ultrahigh-temperature (UHT)-treated milk; UHT skimmed milk, UHT semi-skimmed milk) was investigated.Stainless steel was exposed to three types of milk with a different amount of fat component. Contact angles measurements were used to determine the surface physicochemical properties of substratum treated with the three milk products. The hydrophobicity and electron acceptor properties of stainless steel seem to be decreasing with the amount of fat component present in milk but its electron donor property increase with this component. The ability of S. aureus to adhere to stainless steel treated and untreated with milk was also examined. Treatment with the three types of milk reduces bacterial attachment. On treated substratum, the adhesion extent was affected by the type of milk and consequently by the amount of fat component. The lower and the higher adhesion were obtained when the steel was treated by the UHT semi-skimmed milk and UHT skimmed milk respectively. The correlation between physicochemical properties and S. aureus adhesion show that this latter was controlled by hydrophobicity and electron donor properties.The findings of this work can contribute to develop strategies for prevent S. aureus adhesion on stainless steel and biofilm formation. Also they could be taken into account in cleaning and disinfection procedures.     

Prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk and dairy products

The objectives of this study were to determine the prevalence and antimicrobial resistance of Staphylococcus aureus isolated from raw milk (cow and sheep) and dairy products (traditional cheese and kashk) in Mazandaran Province, Iran. A total of 2650 samples, including 1930 raw milk and 720 dairy products were purchased from retail stores. Out of 2650 samples, S. aureus was detected in 328 samples (12.4%) in which 53 (16.2%) were positive for methicillin-resistant S. aureus. The S. aureus isolates showed resistance to tetracycline (56.1%), followed by penicillin G (47.3%), oxacillin (16.2%), lincomycin (11.9%), clindamycin (11.3%), erythromycin (7.9%), streptomycin (5.8%), cefoxitin (5.5%), kanamycin (4%), chloramphenicol (3.7%), and gentamicin (2.1%). A high frequency of blaZ (46%) and tetM (34.8%) resistance genes was found in S. aureus isolates. The findings of this study revealed consumption of raw milk and dairy products as a potential risk of foodborne infection in this region.     

Inactivation of Staphylococcus spp. strains in whole milk and orange juice using ultra high pressure homogenisation at inlet temperatures of 6 and 20 °C

The objective of this work was to evaluate the inactivation induced by ultra high pressure homogenisation (UHPH) of Staphylococcus aureus ATCC 13565 and Staphylococcus carnosus CECT 4491 inoculated into milk and orange juice considering the effect of inlet temperature of the sample (6 and 20 °C) on the lethality values and on the production of sublethal injuries. Samples of UHT whole milk and UHT orange juice were inoculated at a concentration of approximately 7.0 log (CFU/ml) and pressurized with a dual valve UHPH machine at 300 MPa at the primary homogenising valve and at 30 MPa on the secondary valve. Viable and injured bacterial counts were measured 2 h after UHPH treatment and after 3, 6, and 9 days of storage at 4 °C for milk, and after 3, 6, 9, 12, and 15 days of storage at 4 °C for orange juice. The inlet temperature, the food matrix and the kind of strain influenced significantly (P < 0.05) the lethality level, which was higher for S. aureus in whole milk at an inlet temperature of 20 °C. No sublethal injuries were detected after treatments. The change over time of viable counts for both strains showed a very strong decreasing tendency during the storage at 4 °C for orange juice, while the strain S. carnosus showed a low decreasing tendency and greater resistance when inoculated in milk and pressurized at 6 °C.     

Effects of temperature on the viability,growth and gene profile of Yersinia pseudotuberculosis and Yersinia enterocolitica inoculated in milk

This study was aimed at deciphering the viability and growth of Yersinia pseudotuberculosis and Yersinia enterocolitica in milk at different temperatures, and at identifying the temperature-dependent changes in gene expression in Yersinia. Fresh Yersinia culture was suspended either in pasteurized or in autoclaved milk and subjected to different temperature ranges. Colony forming units (CFU) were determined from inoculated milk after one and two weeks of storage using direct plating technique. In both one and two weeks of storage, growth of Y. pseudotuberculosis and Y. enterocolitica increased significantly at 4 °C and 24 °C (P < 0.05). Furthermore, gene expression profile and DNA microarray analyses were conducted. After one-week storage, the growths of Y. pseudotuberculosis and Y. enterocolitica were optimal at 4 °C and 24 °C. Remarkably, at 37 °C there was no detectable level of CFU from both Yersinia spp inoculated in pasteurized milk, whereas they grew well at 37 °C in autoclaved milk. The NotI-profile of Y. pseudotuberculosis grown at 24 °C generated different banding patterns from other treatment groups when compared to FseI and XbaI-PFGE pulso-type. Microarray interrogation of 4038 genes of Y. pseudotuberculosis revealed that 38 genes were upregulated by ≥8 fold and 237 genes exhibited ≥2 fold downregulation (at 95% significant level) after temperature shift from 4 °C to 24 °C. The findings of this study highlight the survival potential of Y. pseudotuberculosis and Y. enterocolitica in milk under different temperatures and the associated gene expression patterns, which may be important in the processing and safety of milk and dairy products.     

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in retail raw chicken in China

The aim of this study was to determine the prevalence of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) in whole raw chickens in China. A total of 1152 whole chicken samples were collected from three types of retail markets (large supermarket, small supermarket, and farmers market) in six provinces and two national cities in China and screened for the presence of. S. aureus, then the S. aureus isolates were further examined for the presence of MRSA. The overall positive rate for S. aureus was 24.2%. S. aureus prevalence was highest in Shaanxi province (39.6%), followed by Fujian province (31.3%), then Sichuan and Guangdong provinces (26.4% in each province), Shanghai (23.6%), Beijing (19.4%), Henan province (13.9%), the lowest prevalence was found in Guangxi province (13.2%). S. aureus prevalence was significantly different among the six provinces and two national cities (P < 0.05). Among 1152 chicken samples, twenty samples (1.7%) were positive for MRSA. MRSA prevalence was highest in Shaanxi province and Sichuan province (3.5% for each), then Fujian province (2.8%), Guangdong province and Shanghai (2.1% for each); MRSA isolates were not detected from Beijing, Henan province and Guangxi province. The overall S. aureus prevalence in chickens collected from large supermarket, small supermarket, and farmers market were 26.3%, 25%, and 21.4%, respectively, but differences were not significant (P > 0.05). These findings indicate the need for identifying the factors contributing to the contamination at the farm, processing and retail level, and for the implementation of good manufacturing practice to better control the pathogen throughout poultry industry.     

Investigation of the food value chain of ready-to-eat chicken and the associated risk for staphylococcal food poisoning in Tshwane Metropole,South Africa

The objective of the study was to better understand the informal markets for ready-to-eat (RTE) chicken in Tshwane Metropole, Gauteng Province, South Africa, and in particular the links between the formal and informal sector. As part of this, we assessed the risk of a common food poisoning (staphylococcal) through consumption of RTE chicken sold by informal vendors. We used participatory risk assessment, a novel approach to understanding food safety in data scarce environments to collect information. Structured interviews and focus group discussions with informal vendors (n = 237) were conducted to understand poultry value chains for informal RTE chicken, business operation and hygiene practices. Samples (n = 100) of RTE were collected from informal vendors in six major taxi ranks. Staphylococcus aureus counts were determined using 3M™ Petrifilm™ plates. Data collected in this present study plus information obtained from reviewing of literature, were used to develop a stochastic risk model. The number of colonies which were too numerous to count (TNTC) was artificially modeled.A mapping of the informal food value chain revealed that there are four possible value chains and that chicken spilled over from formal to informal markets. The prevalence of S. aureus in RTE chicken samples (44%; 90% CI: 36.1%–52.2%) was high. The mean S. aureus counts in the ready to eat chicken was 10^3.6 (90%CI: 10^3.3–10^3.9), and the risk of purchasing chicken of unsatisfactory quality (>10³ cfu/g) was 32.9% (90%CI: 25.5%–40.4%). The probability of food poisoning due to consumption of RTE chicken contaminated with staphylococcal enterotoxin was estimated to be 1.3% (90% CI: 0%–2.7%). Sensitivity analysis showed that the probability of S. aureus having the enterotoxin gene was the most sensitive parameter for food poisoning. This was followed by S. aureus concentration in RTE chicken and lastly the prevalence of S. aureus in ready-to-eat chicken.This study demonstrates the existence of a strong link between formal and the informal market. In view of the low risk observed, the relevant authorities in Tshwane should continue to support the informal sale of RTE chicken. However, there is still a need for provision of hygiene training to reduce the concentration levels of S. aureus on the RTE chicken, and to promote the sale of safer affordable source of protein for the large urban poor population in South Africa. This will also help secure the opportunities for employment associated with the trade.     

The specific antibacterial effect of the Salvia oil nanoliposomes against Staphylococcus aureus biofilms on milk container

The Staphylococcus aureus (S. aureus) biofilm on container is the main source of microbial contamination in milk. In this study, the nanoliposomes encapsulating Salvia oil (SO) has been prepared. Based on the damage effect of pore-forming toxin on cell membrane, α-toxin secreted from S. aureus has been used to trigger the release of SO from nanoliposomes to achieve antibacterial effect on S. aureus biofilm on milk container. Firstly, the minimum biofilm eradication concentration (MBEC) and biofilms time-dependent killing of SO were tested. The results showed that the MBEC of SO against S. aureus biofilms was 0.2% and SO effectively eradicated the biofilms after treating for 4 h. Subsequently, SO was encapsulated into nanoliposomes in order to increase its stability. The particle size, poly dispersity index (PDI), zeta potential, pH, turbidity, and entrapment efficiency of SO nanoliposomes were analyzed systematically. Gas chromatography–mass spectrometry (GC–MS) has been utilized to observe the controlled release of SO form nanoliposomes incubated with S. aureus. The scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) images have also visually showed that SO nanoliposomes have high anti-biofilm activity against S. aureus biofilms on milk container.     

Pre-processed bovine milk quality in Trinidad: Prevalence and characteristics of bacterial pathogens and occurrence of antimicrobial residues in milk from collection centres

The study was conducted to assess the impact of the changes in the milk collection system in Trinidad (from twice daily collection to once, introduction of chilling facilities to the collection centres and transportation of milk to the processing plant in insulated truck instead of in metal churns at ambient temperature) on the microbial load and antimicrobial residue quality of the milk as well as the temperature and pH of milk, using standard methods. The presence of antimicrobial residues was detected using the Delvo test kit. Of a total of 266 milk samples from churns, the mean ± sd temperature and log₁₀ ± sd TAPC per ml was 20.36 ± 7.91 °C and 6.3 ± 1.09 respectively. For 20 milk samples from the chillers, the mean temperature and log₁₀ ± sd TAPC per ml was 15.10 ± 2.73 °C and 7.04 ± 0.33 respectively compared with corresponding values for 36 samples collected from the truck, 11.64 ± 4.22 °C and 7.11 ± 0.62 respectively (P < 0.05; X²). The mean TAPC, staphylococcal and E. coli counts per ml of milk from churns were significantly (P < 0.05; X²) higher for milk at low temperature (0–20 °C) compared with milk at high temperature (>30 °C). Eight (4.2%) of 192 milk samples tested contained antimicrobial residues. Of 168 S. aureus isolates tested, 24 (14.3%) were enterotoxigenic while 53 (45.3%) of 117 isolates tested exhibited resistance to various antimicrobial agents while of 386 isolates of E. coli tested, 3 (0.8%) were O157 strain and 129 (64.5%) of 200 isolates exhibited resistance to antimicrobial agents. It was concluded that despite the changes, the microbial load of milk was still high suggesting poor sanitary practices at the farm level. The detection of antimicrobial residues agents coupled with toxigenic S. aureus and E. coli isolates could pose health hazards to consumers.     

Investigation into the antibacterial behavior of suspensions of magnesium oxide nanoparticles in combination with nisin and heat against Escherichia coli and Staphylococcus aureus in milk

The antibacterial activities of magnesium oxide nanoparticles (MgO NP) alone or in combination with nisin against Escherichia coli and Staphylococcus aureus were investigated. Synergistic antibacterial effects existed and at lower levels of nisin when compared to when nisin was used alone. Also the antibacterial activities of MgO NP in combination with other antimicrobials (nisin and heat) against E. coli and S. aureus were investigated in milk. A synergistic effect of MgO NP in combination with nisin and heat was observed as well. Scanning electron microscopy was used to characterize the morphological changes of E. coli after antimicrobial treatments. It was revealed that MgO NP treatments in combination with nisin distort and damage the cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells. This is the first report describing the antibacterial activity of MgO NPs and nisin in milk. It leads the way to development of treatment combinations which could result in a decrease in pasteurisation temperatures and the level of MgO NP required for pasteurising milk and maintaining pathogen control.     

Bactericidal effects of Cinnamon cassia oil against bovine mastitis bacterial pathogens

Organic food production is expanding rapidly. However, this industry is hampered by the lack of effective antimicrobial agents which can be used in organic food production. This study examined the antimicrobial activity of Cinnamon cassia oil against major pathogens causing bacterial bovine mastitis, its miscibility in milk and possible antimicrobial mechanisms. C. cassia oil had inhibitory activity against all tested pathogen isolates from bovine mastitis. We conducted disk diffusion assay and found that discs with 20 μL of 2% (v/v) C. cassia oil solution resulted in inhibition zones of 29.6, 19.1, 27.0, 33.3 and 30.7 mm for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus xylosus and Escherichia coli 29, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C. cassia oil was 0.00625% and 0.025% (v/v) for S. hyicus, 0.025% and 0.10% (v/v) for E. coli 29, and 0.0125% and 0.05% for S. aureus, S. epidermidis and S. xylosus, respectively. We selected two common mastitis pathogens, a representative S. aureus isolate and E. coli 29 for further analyses. Based on time-kill assay in LB broth with 0.15% agar, 2MBC of C. cassia oil generated bactericidal effects on S. aureus and E. coli 29 within 30 min, and 4MBC caused 6 log reduction of S. aureus and E. coli 29 within 30 min. In milk, C. cassia oil at 4MBC reduced ∼6.0 Log₁₀ CFU/ml of S. aureus and E. coli 29 to undetectable level within 8 h. Using propidium iodide staining, we observed membrane damage on both S. aureus and E. coli 29 cells during incubation with C. cassia oil. In addition, C. cassia oil treatment at MIC impaired membrane integrity of E. coli and S. aureus, which was followed by a decrease in ATP synthesis. Bacterial extracellular signaling quorum sensing orchestrates important events related to bacterial pathogeneses through excreting autoinducer (AI). Sub-inhibitory concentration of C. cassia oil repressed AI-2, a universal signal molecule mediating quorum sensing, production in S. aureus and E. coli 29 isolates. Collectively, our data show that C. cassia oil provides an exciting potential to be used as an alternative antimicrobial for bovine mastitis in organic dairy farms.     

Detection of methicillin-resistant Staphylococcus aureus in dairy cow farms

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens causing nosocomial infections worldwide. Animal-associated MRSA hazard has been recently identified, but less information is currently available regarding MRSA in cattle. The aim of this study was to estimate the presence of MRSA in samples of bulk milk, environmental dust, conjunctival and nasal swabs of workers obtained from thirty dairy cow farms. A total of 200 S. aureus strains were identified using phenotypic and molecular approaches. Three other species (Staphylococcus epidermidis, Staphylococcus xylosus and Staphylococcus saprophyticus) were found. In five S. aureus strains isolated from environmental dust and one S. epidermidis strain derived from human samples, mecA gene was detected showing a specific fragment at 527 bp. Moreover, 66 S. aureus strains were distinguished by susceptibility to 15 antimicrobial agents. The highest resistance profile was ascribed to ampicillin, amoxicillin and penicillin G, both in workers and bulk milk samples. Generally, a multiple resistance to 4 up to 10 antibiotics was detected. S. aureus mecA⁺ strains and S. epidermidis mecA⁺ strain showed multiple resistance to 13 and 11 antibiotics, respectively. The obtained results suggested that the low number of MRSA strains, probably of human origin, was due to the appropriate hygienic practices adopted by the dairy cow farms.     

Modelling survival behaviour of Salmonella enterica ser. Enteritidis,Typhimurium and Tennessee on table eggs during storage at different temperatures

Quantitative risk assessment studies on the health risk of Salmonella due to consumption of contaminated table eggs are based on the assumption that Salmonella is inside the egg and that the pathogen belongs to serovar Enteritidis. However different serovars of Salmonella may contaminate the surface of table eggs and spread to other foods at consumer's kitchen due to improper food handling. In the present study the survival behaviour of one strain each from Salmonella enterica serovars Enteritidis, Typhimurium and Tennessee on table egg surface during storage at 4, 8, and 20 °C have been described. Besides, in those cases observed data were subjected for modelling; linear, log-linear tail and Weibull models were compared in terms of model fitting and model performance. Overall, in most cases, inactivation kinetics presented a linear trend on Salmonella behaviour so that Weibull and linear models adequately described observed data. Regarding log-linear tail models, though they presented a better fitting, their adequacy could not be assessed given the lack of data in the tail region. Regarding storage temperatures, 4 °C was predicted to be the most inhibitory temperature for table eggs externally contaminated by a strain of S. enterica serovar Enteritidis. After 28 days of storage, a reduction of 4 log₁₀ cfu/g of eggshell on the S. enterica ser. Enteritidis load was registered at 4 °C. S. enterica ser. Typhimurium and Tennessee showed higher survival rates at all tested temperatures. The results highlighted the importance of keeping constant the storage temperature of table eggs in order to reduce the risk of S. enterica ser. Enteritidis contaminating the surface of table eggs. However this temperature might not be the optimal one in view of S. enterica serovars other than Enteritidis.     

High hydrostatic pressure treatment applied to model cheeses made from cow’s milk inoculated with Staphylococcus aureus

Pasteurized milk was inoculated with two strains of Staphylococcus aureus (CECT4013 or ATCC13565) and used to elaborate soft-curd cheeses with approximately 7.5-log CFU/g of S. aureus. Cheeses were submitted to 10 min high hydrostatic pressure (HHP) treatments of 300, 400 or 500 MPa at 5 °C or 20 °C. Staphylococcus enterotoxin (SE) was evaluated in cheeses containing ATCC13565. Counts of S. aureus were measured after HHP treatment (day 1) and after 2, 15 and 30 days ripening at 8 °C. Inactivation increased with pressure and storage time, but was similar for both treatment temperatures. Maximum S. aureus reductions were achieved after 30 days ripening for samples treated at 500 MPa and 5 °C: 6.0 ± 0.1 and 4.7 ± 0.5-log CFU/g for CECT4013 and ATCC13565, respectively. However, SE was detected in all cheese samples containing ATCC13565 before and after HHP and after 30 days ripening.     

Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10⁷ CFU/mL of dead Salmonella Typhimurium, Shigella sonnei, and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium, S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10¹ CFU/mL or 10¹ CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus.     

Suitability of ccRSM as a tool to predict inactivation and its kinetics for Escherichia coli,Staphylococcus aureus and Pseudomonas fluorescens in homogenized milk treated by manothermosonication (MTS)

The application of ultrasound (US) technology (i.e. sonication) has been used as an alternative to thermal technologies to reduce the microbial population of foods and beverages. This approach can be used on its own or combined with pressure and temperature (i.e. manothermosonication (MTS)). Microbial inactivation kinetics by sonication remains unclear. In the present study, a central composite response surface model (ccRSM) was used in order to study the effects of temperature (20–52 °C), acoustic intensity (60–120 W/cm²) and treatment time (40–240 s) at a constant pressure (225 kPa) by MTS processing on microbial inactivation. Reductions of up to c. 1.6 log CFU/ml were achieved for Escherichia coli and Pseudomonas fluorescens. Lower inactivation values were reported for Staphylococcus aureus (1.05 log CFU/ml). These inactivation values were achieved using conditions of 36 °C, 90 W/cm² and 240 s. Inactivation of E. coli by MTS (R² = 0.90) was described by an exponential curve, whereas inactivation of S. aureus and P. fluorescens by MTS (R² ≥ 0.73) were described by a linear trend. The coefficient of determination values were obtained following validation between the theoretical model and the experimental values. Statistical analysis showed that treatment time was the factor with greatest influence on microbial inactivation for all three microorganisms. The present study confirms the suitability of using ccRSM as a tool for investigating and predicting the inactivation of E. coli, S. aureus and P. fluorescens as a function of temperature, acoustic power and treatment time, and may also be useful for predicting inactivation of other microorganisms commonly found in raw milk.     

Comparing the pulsed electric field resistance of the microorganisms in grape juice: Application of the Weibull model

Pulsed electric field (PEF) technology is a nonthermal food preservation technology that is based on the use of electric field to eradicate microorganisms in foods. In present study, the PEF resistance of three strains of microorganisms (Staphyloccocus aureus, Escherichia coli DH5α and Saccharomyces cerevisiae) in grape juice was investigated by applying PEF treatments ranging from 9 kV/cm to 27 kV/cm and from 34 μs to 275 μs at an initial treatment temperature of 40 °C. The temperature measurement at particular locations in the processing line showed a general trend for all the composites: greater temperature increasing as the electric field strength and treatment time values increased. In all experimental conditions used in this investigation temperature increments were always lower than 8.8 °C. The obtained survival curves indicated that the inactivation of three strains increased with electric field strength and treatment time, or specific energy. The S. cerevisiae exhibited the least resistance to PEF treatments, S. aureus the second, E. coli DH5α the third. The experimental kinetics of inactivation by PEF of these microorganisms has been described by mathematical equations based on the modified Weibull model, using treatment time and specific energy as the control parameters. Both R² and RMSE indicated a slightly higher degree of correlation between experimental and fitted values was achieved for models in terms of specific energy. Finally, the predicted inactivation curves from the established model were validated to evaluate the performance of the model using a total of 36 datasets for three microorganisms. Models built in terms of specific energy showed better accuracy than those in terms of treatment time. Whether treatment time or specific energy was used as control parameter of the model, all the bias factors ranged within the proposed acceptable limits. Overall, 80.6% and 85% of the mean prediction errors for above two models were in the acceptable prediction zone, more than 70% of which was considered as a threshold for a simulation model to provide acceptable predictions. The validation results indicated that the Weibull models were safe for predicting inactivation curves of S. aureus, E. coli DH5α and S. cerevisiae by PEF in grape juice. Therefore, the developed tertiary models were useful for assessing the lethal effects of different parameters on PEF treatment results and evaluating the relation between cost and benefit of a potential implementation of PEF technology in industrial production processes.