Comparison of SYBR® Green qPCR assay and plate count method to describe growth of Weissella viridescens and Leuconostoc mesenteroides in pure and mixed cultivation.

The current study was conducted to statistically compare the SYBR® Green quantitative polymerase chain reaction (qPCR) assay and the conventional plate counting (PC) method to construct growth curves of a cocktail of Weissella viridescens in pure culture under different isothermal storage conditions (4, 8, 14, and 30 °C) and in mixed culture with Leuconostoc mesenteroides at 8 °C. The efficiency and specificity of the qPCR standard curves were confirmed, and both methods were adequate to quantify the growth kinetics of W. viridescens at all isothermal temperatures, demonstrating a good correlation and agreement. The efficiencies of the standard curves varied between 98% and 102%. The SYBR® Green qPCR assay was also able to differentiate the growth curves of W. viridescens and L. mesenteroides in the mixed culture at 8 °C. Additionally, the SYBR® Green qPCR method was considered a faster and more sensitive alternative to construct growth curves under different isothermal conditions and differentiate morphologically similar lactic acid bacteria. Overall, the results suggest that the SYBR® Green qPCR method is a reliable and efficient tool to study microbial growth kinetics in pure and mixed cultures.

Modeling the growth dependence of Streptococcus thermophilus and Lactobacillus bulgaricus as a function of temperature and pH.

The growth of the lactic acid bacteria (LAB), Streptococcus thermophilus and Lactobacillus bulgaricus, widely used for yogurt production, results in acid production and the reduction of the milk [Formula: see text]. Industrial processes can show temperature ([Formula: see text]) changes due to the large scale of the equipment. As [Formula: see text] and [Formula: see text] affect the LAB growth, this study aimed to model the dependence of S. thermophilus and L. bulgaricus as a function of temperature and pH and to estimate and internally validate their growth parameters and confidence intervals with different modeling approaches. Twenty-four datasets regarding the growth kinetics of S. thermophilus and L. bulgaricus were used for estimating the kinetic parameters for each pure culture. The classical Baranyi and Roberts (sigmoidal) primary and Rosso and coworkers (cardinal parameter) secondary models successfully described the experimental data. The one-step modeling approach showed better statistical results than the two-step approach. The values of eight growth parameters ([Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text]) for each culture estimated from the fitting with the one-step approach and the Monte-Carlo-based approach were similar. Low averaged root-mean-squared errors ([Formula: see text]) (0.125 and 0.090 log CFU/mL) and percent discrepancy factor [Formula: see text] ([Formula: see text] and [Formula: see text]) values for S. thermophilus and L. bulgaricus were obtained in the internal model validation, reinforcing the predictive ability of the model.

Microcalorimetric growth behavior of E. coli ATCC 25922 in an MCDSC.

Isothermal microcalorimetry can provide a general analytical tool for the characterization of bacterial growth. Methodologies and equipment have been studied to expand the application and disseminate the use of the technique. The MCDSC is a microcalorimeter capable of measuring in the range of 0.2 µW that can operate at a temperature range of -20 to 140 °C or under isothermal conditions. Here, we present the first investigation of MCDSC for E. coli growth with the Baranyi and Roberts modeling application. This study presented the calorimetric E. coli fingerprint at MCDSC and compares it with the plate count technique, giving the data more biological meaning. The calorimeter was able to accurately detect growth metabolism and discriminate E. coli at different inoculum densities. Additionally, the MCDSC can offer a new point of view for evaluating microbial growth, such as the significant reduction in error due to dispersed data by the viable counting method.

Modeling the effect of Croton blanchetianus Baill essential oil on pathogenic and spoilage bacteria.

This study aimed to evaluate and model the antimicrobial action of different concentrations of Croton blanchetianus essential oil (CBEO) on the behavior of six bacterial species in vitro. CBEO extraction was performed by hydrodistillation and characterized by CG-MS. CBEO solutions in culture media were tested at 0.90, 1.80, 2.71, and 4.51 mg of CBEO/mL, against foodborne bacteria: pathogenic bacteria (Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis at 35 °C), a non-pathogenic Escherichia coli (at 35 °C), and spoilage bacteria (Weissella viridescens and Leuconostoc mesenteroides at 30 °C). The CBEO major compounds were eucalyptol, α-pinene, sativene, E-caryophyllene, bicyclogermacrene, and spatulenol. Baranyi and Roberts (growth) and Weibull (inactivation) primary models, along with power and hyperbolic secondary models, were able to describe the data. CBEO inactivated L. monocytogenes, S. aureus, L. mesenteroides and W. viridescens at all applied concentrations. CBEO did not inactivate S. Enteritidis and E. coli, but their growth rates were reduced.

Modelling the inactivation, survival and growth of Salmonella enterica under osmotic stress considering inoculum phase and serotype.

AIMS: This study evaluated the behaviour of the Salmonella enterica serotypes in osmotically stressful BHI broth (0.940 ≤ aw  ≤ 0.960), assessing inoculum from two stages of the bacterial life cycle (exponential and stationary) and two temperatures (25°C and 35°C). METHODS AND RESULTS: Four S. enterica serotypes (Typhimurium, Enteritidis, Heidelberg and Minnesota) were grown in stressful BHI at 25°C. A mathematical model was proposed for describing the total microbial count as the sum of two subpopulations, inactivating and surviving-then-growing. When submitted to aw of 0.950 and 0.960, all strains showed a decreased count, followed by a period of unchanged count and then exponential growth (Phoenix Phenomenon). Strains inoculated at aw  = 0.940 and 0.945 showed inactivation kinetics only. Cells cultivated at 25°C and inoculated from the exponential phase were the most reactive to the osmotic stress, showing a higher initial population reduction and shorter adaptation period. The proposed model described the inactivation data and the Phoenix Phenomenon accurately. CONCLUSIONS: The results quantified the complex response of S. enterica to the osmotic environment in detail, depending on the inoculum characteristic and serotype evaluated. SIGNIFICANCE AND IMPACT OF STUDY: Quantifying these differences is truly relevant to food safety and improves risk analysis.

Assessment of the lipid oxidation, color and sensorial acceptance of fresh sausage formulated with potassium bixinate as a substitute for sodium nitrite and carmine.

This work aimed to evaluate the lipid oxidation, color, and sensory acceptance of sausages formulated with potassium bixinate (BP) as a substitute for carmine (Car) and sodium nitrite (SNi). Six different treatments (CT-control; CA-40 ppm Car; NIT-150 ppm SNi; C/N-40 ppm Car, and 150 ppm SNi; C/N/B-20 ppm Car, 75 ppm SNi, and 250 ppm BP; and BIX-500 ppm BP) were evaluated along 15 days. BP provided significantly lower levels of lipid oxidation to the samples (C/N/B and BIX) when compared to CT and significantly equal to CA, NIT, and C/N after 15 days (both p < 0.05). BP significantly increased the red (a*) and yellow (b*) intensity of sausages (p < 0.05), providing the highest values in both raw and roasted samples. Sausages formulated with BP did not differ in preference in visual analysis (p > 0.05), and both roasted were preferred over CT (p < 0.05). Although sausages formulated with carmine had better visual acceptance, BIX and C/N did not differ significantly (p > 0.05) after five days. BP was identified in taste analysis; however, it was preferred in the ordination analysis (both p < 0.01). Therefore, the replacement of SNi and Car by BP showed to be a viable alternative.

Modeling Salmonella spp. inactivation in chicken meat subjected to isothermal and non-isothermal temperature profiles.

Salmonella genus has foodborne pathogen species commonly involved in many outbreaks related to the consumption of chicken meat. Many studies have aimed to model bacterial inactivation as a function of the temperature. Due to the large heterogeneity of the results, a unified description of Salmonella spp. inactivation behavior is hard to establish. In the current study, by evaluating the root mean square errors, mean absolute deviation, and Akaike and Bayesian information criteria, the double Weibull model was considered the most accurate primary model to fit 61 datasets of Salmonella inactivation in chicken meat. Results can be interpreted as if the bacterial population is divided into two subpopulations consisting of one more resistant (2.3% of the total population) and one more sensitive to thermal stress (97.7% of the total population). The thermal sensitivity of the bacteria depends on the fat content of the chicken meat. From an adapted version of the Bigelow secondary model including both temperature and fat content, 90% of the Salmonella population can be inactivated after heating at 60 °C of chicken breast, thigh muscles, wings, and skin during approximately 2.5, 5.0, 9.5, and 57.4 min, respectively. The resulting model was applied to four different non-isothermal temperature profiles regarding Salmonella growth in chicken meat. Model performance for the non-isothermal profiles was evaluated by the acceptable prediction zone concept. Results showed that >80% of the predictions fell in the acceptable prediction zone when the temperature changes smoothly at temperature rates lower than 20 °C/min. Results obtained can be used in risk assessment models regarding contamination with Salmonella spp. in chicken parts with different fat contents.

Modeling the inactivation of Aspergillus fischeri and Paecilomyces niveus ascospores in apple juice by different ultraviolet light irradiances.

The present work aimed to evaluate and to model the influence of UV-C light treatments with different irradiances (6.5, 13, 21, and 36 W/m2) on Aspergillus fischeri and Paecilomyces niveus ascospores inactivation in clarified apple juice. Approximately 5.0 and 6.0 log CFU/mL spores of P. niveus and A. fischeri, respectively, were suspended in 30 mL of clarified apple juice (pH 3.8, 12 ± 0.1°Brix) and exposed to UV-C light at different irradiances (as above) and exposure times (0 to 30 min). The first-order biphasic model was able to describe the experimental data with good statistical indices (RMSE = 0.296 and 0.308, R2 = 0.96 and 0.98, for P. niveus and A. fischeri respectively). At the highest irradiance level tested (36 W/m2), the UV-C light allowed the reduction of 5.7 and 4.2 log-cycles of A. fischeri and P. niveus ascospores, respectively, in approximately 10 min. P. niveus was the most UV-C resistant mould. The results showed that, to a defined UV-C fluence, a change in the level of either time or UV-C irradiance did not affect the effectiveness of UV-C light for A. fischeri and P. niveus inactivation. Thus, the modeling of the inactivation as a function of the UV-C fluence allowed the estimation of the primary model parameters with all experimental data and, consequently, no secondary models were needed. The model parameters were validated with experiments of variable UV-C fluences. Accordingly, experimental results allowed to conclude that UV-C treatment at the irradiances tested is a promising application for preventing A. fischeri and P. niveus spoilage of juices.

A mathematical modeling approach to the quantification of lactic acid bacteria in vacuum-packaged samples of cooked meat: Combining the TaqMan-based quantitative PCR method with the plate-count method.

The TaqMan-based quantitative Polymerase Chain Reaction (qPCR) method and the Plate Count (PC) method are both used in combination with primary and secondary mathematical modeling, to describe the growth curves of Leuconostoc mesenteroides and Weissella viridescens in vacuum-packaged meat products during storage under different isothermal conditions. Vacuum-Packaged Morcilla (VPM), a typical cooked blood sausage, is used as a representative meat product, with the aim of improving shelf-life prediction methods for those sorts of meat products. The standard curves constructed by qPCR showed good linearity between the cycle threshold (CT) and log10 CFU/g, demonstrating the high precision and the reproducible results of the qPCR method. The curves were used for the quantification of L. mesenteroides and W. viridescens in artificially inoculated VPM samples under isothermal storage (5, 8, 13 and 18 °C). Primally, both the qPCR and the PC methods were compared, and a linear regression analysis demonstrated a statistically significant linear correlation between the methods. Secondly, the Baranyi and Roberts model was fitted to the growth curve data to estimate the kinetic parameters of L. mesenteroides and W. viridescens under isothermal conditions, and secondary models were used to establish the dependence of the maximum specific growth rate on the temperature. The results proved that primary and secondary models were adequate for describing the growth curves of both methods in relation to both bacteria. In conclusion, the results of all the experiments proved that the qPCR method in combination with the PC method can be used to construct microbial growth kinetics and that primary and secondary mathematical modeling can be successfully applied to describe the growth of L. mesenteroides and W. viridescens in vacuum-packaged morcilla and, by extension, other cooked meat products with similar characteristics.

Modeling the effect of oregano essential oil on shelf-life extension of vacuum-packed cooked sliced ham.

The present study modeled the effect of oregano essential oil, as an antimicrobial agent, on the shelf-life of vacuum-packed cooked sliced ham, based on the growth of lactic acid bacteria natural microbiota under isothermal conditions. The bacterial growth in ham without oregano essential oil (control) and with 0.4% oregano essential oil (v/w) was evaluated at five different temperatures (6, 12, 15, 20 and 25°C). Baranyi and Roberts, and modified Gompertz primary models were fitted to microbial growth curves. Square Root and Exponential secondary models were fitted to µmax parameter data. The addition of oregano essential oil increased lag phase, decreased growth rates and extended shelf-life of ham for all temperatures (at 6°C extended for, at least, 30days when compared to control). Statistical indexes showed that Baranyi and Roberts, and Exponential were the primary and secondary models, respectively, that better fit to the data. Thus, oregano essential oil showed a good antimicrobial effect and extended the ham shelf-life.

Modelling the growth of lactic acid bacteria at different temperatures

ABSTRACT Mathematical models are widely used to predict the shelf life of foods. Lactic acid bacteria (LAB), particularly Lactobacillus plantarum, Weissella viridescens and Lactobacillus sakei, are the main spoilage bacteria of refrigerated, vacuum-packed meat products, stored in modified atmosphere, and their growth determines the shelf life length of these products. The objective of this study was to model the growth of L. plantarum, W. viridescens and L. sakei under different isothermal cultivation conditions and establish secondary models to describe the effect of temperature on the growth parameters of these bacteria. The LAB growth was evaluated in culture medium at temperatures of 4, 8, 12, 16, 20 and 30 ºC. The fit of Baranyi and Roberts (BAR) and Gompertz (GO) primary models to the growth curves of LAB was compared by statistical indices, in which the BAR model showed slightly better fits to the experimental data. The BAR growth parameters were used to establish the secondary models, µmax and Nmax were established for the three LAB. The power model described the influence of temperature on the parameter λ for L. plantarum, and other bacteria showed no lag phase. The growth of LAB was strongly influenced by storage temperature and the obtained models allow predicting the growth of these bacteria within the temperature range from 4 to 30 ºC.

Optimal experimental design for improving the estimation of growth parameters of Lactobacillus viridescens from data under non-isothermal conditions.

In predictive microbiology, the model parameters have been estimated using the sequential two-step modeling (TSM) approach, in which primary models are fitted to the microbial growth data, and then secondary models are fitted to the primary model parameters to represent their dependence with the environmental variables (e.g., temperature). The Optimal Experimental Design (OED) approach allows reducing the experimental workload and costs, and the improvement of model identifiability because primary and secondary models are fitted simultaneously from non-isothermal data. Lactobacillus viridescens was selected to this study because it is a lactic acid bacterium of great interest to meat products preservation. The objectives of this study were to estimate the growth parameters of L. viridescens in culture medium from TSM and OED approaches and to evaluate both the number of experimental data and the time needed in each approach and the confidence intervals of the model parameters. Experimental data for estimating the model parameters with TSM approach were obtained at six temperatures (total experimental time of 3540h and 196 experimental data of microbial growth). Data for OED approach were obtained from four optimal non-isothermal profiles (total experimental time of 588h and 60 experimental data of microbial growth), two profiles with increasing temperatures (IT) and two with decreasing temperatures (DT). The Baranyi and Roberts primary model and the square root secondary model were used to describe the microbial growth, in which the parameters b and Tmin (±95% confidence interval) were estimated from the experimental data. The parameters obtained from TSM approach were b=0.0290 (±0.0020) [1/(h0.5°C)] and Tmin=-1.33 (±1.26) [°C], with R2=0.986 and RMSE=0.581, and the parameters obtained with the OED approach were b=0.0316 (±0.0013) [1/(h0.5°C)] and Tmin=-0.24 (±0.55) [°C], with R2=0.990 and RMSE=0.436. The parameters obtained from OED approach presented smaller confidence intervals and best statistical indexes than those from TSM approach. Besides, less experimental data and time were needed to estimate the model parameters with OED than TSM. Furthermore, the OED model parameters were validated with non-isothermal experimental data with great accuracy. In this way, OED approach is feasible and is a very useful tool to improve the prediction of microbial growth under non-isothermal condition.

Modeling the growth of Lactobacillus viridescens under non-isothermal conditions in vacuum-packed sliced ham.

Lactic acid bacteria (LAB) are responsible for spoiling vacuum-packed meat products, such as ham. Since the temperature is the main factor affecting the microbial dynamic, the use of mathematical models describing the microbial behavior into a non-isothermal environment can be very useful for predicting food shelf life. In this study, the growth of Lactobacillus viridescens was measured in vacuum-packed sliced ham under non-isothermal conditions, and the predictive ability of primary (Baranyi and Roberts, 1994) and secondary (Square Root) models were assessed using parameters estimated in MRS culture medium under isothermal conditions (between 4 and 30°C). Fresh ham piece was sterilized, sliced, inoculated, vacuum-packed, and stored in a temperature-controlled incubator at five different non-isothermal conditions (between 4 and 25°C) and one isothermal condition (8°C). The mathematical models obtained in MRS medium were assessed by comparing predicted values with L. viridescens growth data in vacuum-packed ham. Its predictive ability was assessed through statistical indexes, with good results (bias factor between 0.95 and 1.03; accuracy factor between 1.04 and 1.07, and RMSE between 0.76 and 1.33), especially in increasing temperature, which predictions were safe. The model parameters obtained from isothermal growth data in MRS medium enabled to estimate the shelf life of a commercial ham under non-isothermal conditions in the temperature range analyzed.

Modeling Growth Kinetic Parameters of Salmonella Enteritidis SE86 on Homemade Mayonnaise Under Isothermal and Nonisothermal Conditions.

During the last decade, a specific strain of Salmonella Enteritidis (named SE86) has been identified as the major etiological agent responsible for salmonellosis in the State of Rio Grande do Sul, Southern Brazil, and the main food vehicle was homemade mayonnaise (HM). This study aimed to model the growth prediction of SE86 on HM under isothermal and nonisothermal conditions. SE86 was inoculated on HM and stored at 7, 10, 15, 20, 25, 30, and 37°C. Growth curves were built by fitting data to the Baranyi's DMFit, generating r(2) values greater than 0.98 for primary models. Secondary model was fitted with Ratkowsky equation, generating r(2) and root mean square error values of 0.99 and 0.016, respectively. Also, the growth of SE86 under nonisothermal conditions simulating abuse temperature during preparation, storage, and serving of HM was studied. Experimental data showed that SE86 did not grow on HM at 7°C for 30 days. At 10°C, no growth was observed until approximately 18 h, and the infective dose (assumed as 10(6) CFU/g) was reached after 8.1 days. However, the same numbers of SE86 were attained after 6 hours at 37°C. Experimental data demonstrated shorter lag times than those generated by ComBase Predictive Models, suggesting that SE86 is very well adapted for growing on HM. SE86 stored under nonisothermal conditions increased population to reach about 10(6) CFU/g after approximately 30 hours of storage. In conclusion, the developed model can be used to predict the growth of SE86 on HM under various temperatures, and considering this pathogen, HM can be produced if safe eggs are used and HM is stored below 7°C.

Modeling the growth of Byssochlamys fulva and Neosartorya fischeri on solidified apple juice by measuring colony diameter and ergosterol content.

Byssochlamys fulva and Neosartorya fischeri are heat-resistant fungi which are a concern to food industries (e.g. apple juice industry) since their growth represents significant economic liabilities. Although the most common method used to assess fungal growth in solid substrates is by measuring the colony's diameter, it is difficult to apply this method to food substrates. Alternatively, ergosterol contents have been used to quantify fungal contamination in some types of food. The current study aimed at modeling the growth of the heat-resistant fungi B. fulva and N. fischeri by measuring the colony diameter and ergosterol content, fitting the Baranyi and Roberts model to the results, and finally establishing a correlation between the parameters of the two analytical methods. Whereas the colony diameter was measured daily, the quantification of ergosterol was performed when the colonies reached diameters of 30, 60, 90, 120 and 150 mm. Results showed that B. fulva and N. fischeri were able to grow successfully on solidified apple juice at 10, 15, 20, 25 and 30 °C, and the Baranyi and Roberts model showed good ability to describe growth data. The correlation curves between the parameters of colony diameter and ergosterol content were obtained with satisfactory statistical indexes.

Modeling the Growth of Byssochlamys fulva on Solidified Apple Juice at Different Temperatures

The aim of this study was to establish primary and secondary models to describe the growth kinetics of Byssochlamys fulva on solidified apple juice at different temperatures. B. fulva was inoculated on solidified apple juice at 10, 15, 20, 25 and 30 °C. Linear-with-breakpoint, Baranyi and Roberts, and Huang primary models (without upper asymptote) were fitted to the data, and they showed good ability to describe the growth kinetics. B. fulva showed longer adaptation time on apple juice than on culture medium, but growth rates were similar as reported in the literature. The dependence of µmax and λ parameters on temperature was described with Square Root and Arrhenius-Davey secondary models, respectively. These models were important to establish process/storage conditions and apple juice shelf life.

Assessing the prediction ability of different mathematical models for the growth of Lactobacillus plantarum under non-isothermal conditions.

Mathematical models taking temperature variations into account are useful in predicting microbial growth in foods, like meat products, for which Lactobacillus plantarum is a mesophilic and one of the main spoiling bacterium. The current study assessed the ability of the main primary models and their non-isothermal versions to predict L. plantarum growth under constant and variable temperature. Experimental data of microbial growth were obtained in MRS medium under isothermal conditions (4, 8, 12, 16, 20, and 30°C) which were used to obtain the secondary models. The experimental data under non-isothermal conditions (periodically oscillating temperature between the plateaus 4-12, 5-15, and 20-30°C) were used to validate the non-isothermal models. The bias factors indicated that all assessed models provided safe predictions of the microorganism growth at the non-isothermal conditions. Overall, despite the very good performance of the primary models (isothermal), none of the models was able to predict with accuracy the L. plantarum growth under temperature variations, mainly when the temperature range was close to refrigeration temperature. Incorporating the complex microbial adaptation mechanisms into the predictive models is a challenge to be overcome.