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 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.

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.

Effect of soluble solids concentration on Neosartorya fischeri inactivation using UV-C light.

Ascospores of Neosartorya fischeri are heat-resistant and can survive thermal commercial treatments normally applied to the juices, as apple juice. Non-thermal processing of food such as exposure to ultraviolet light (UV-C) is reported to induce minimal quality changes while reduces microbial load. The main objective of this study was to determine the effect at different soluble solids concentration (12, 25, 30, 40, 50, 60 and 70 °Brix) on N. fischeri ascospores inactivation in apple juice, using UV-C light intensity (38 W/m2). Weibull model was fitted to experimental data. Then, a secondary model was used to describe how the inactivation kinetic parameters varied with the changes in soluble solids concentration. Results showed that the UV-C light had influence on N. fischeri ascospores inactivation in apple juice even at the highest soluble solids concentrations used, reaching approximately 4 log reductions at all concentrations used. The inactivation parameters, obtained by Weibull model, were δ (dose for the first decimal reduction) and p (the shape factor). Exponential model was chosen to describe the influence of soluble solids concentration on δ and p parameters. It can be concluded that UV-C light is a promising treatment with a drastic impact on the loads of N. fischeri, especially when low soluble solids concentration is used and a model was obtained to describe Brix effect.

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.

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.

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.

Citrinin mycotoxin recognition and removal by naked magnetic nanoparticles.

Citrinin is a nephrotoxic mycotoxin which can be synthesized by Monascus mold during the fermentation process in foods. Monascus, generally described as red mold, is a red-pigmented filamentous fungus attracting a great interest for the production of natural dyes and cholesterol-lowering statins. We individuated a specie of Monascus producing high amount of natural dyes. However, this high pigmentation was correlated with the production of citrinin. Peculiar magnetic nanoparticles, synthesized in-house and called "Surface Active Maghemite Nanoparticles" (SAMNs), are proposed as an efficient and reliable mean for citrinin removal from Monascus treated foods. The nanomaterial efficiency for citrinin binding was proved on Monascus suspensions, and SAMN@citrinin complex was characterized by MÓ§ssbauer spectroscopy and magnetization measurements, showing that SAMNs resulted structurally and magnetically well conserved after citrinin binding. SAMNs are excellent and stable magnetic nano-carrier for toxin removal, which can be applied in food industry.

Poly(3-Hydroxybutyrate) Production in Repeated fed-Batch with Cell Recycle Using a Medium with low Carbon Source Concentration.

Among approaches applied to obtain high productivity and low production costs in bioprocesses are high cell density and the use of low cost substrates. Usually low cost substrates, as waste/agroindustrial residues, have low carbon concentration, which leads to a difficulty in operating bioprocesses. Real time control of process for intracellular products is also difficult. The present study proposes a strategy of repeated fed-batch with cell recycle to attain high cell density of Cupriavidus necator and high poly(3-hydroxybutyrate) (P(3HB)) productivity, using a substrate with low carbon source concentration (90 g l(-1)). Also, the use of the oxygen uptake rate data was pointed out as an on line solution for process control, once P(3HB) is an intracellular product. The results showed that total biomass (X), residual biomass (Xr) and P(3HB) values at the end of the culture were 61.6 g l(-1), 19.3 g l(-1) and 42.4 g l(-1) respectively, equivalent to 68.8 % of P(3HB) in the cells, and P(3HB) productivity of 1.0 g l(-1) h(-1). Therefore, the strategy proposed was efficient to achieve high productivity and high polymer content from a medium with low carbon source concentration.

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.

Survey of molds, yeast and Alicyclobacillus spp. from a concentrated apple juice productive process.

Bacteria and molds may spoil and/or contaminate apple juice either by direct microbial action or indirectly by the uptake of metabolites as off-flavours and toxins. Some of these microorganisms and/or metabolites may remain in the food even after extensive procedures. This study aim to identify the presence of molds (including heat resistant species) and Alicyclobacillus spp., during concentrated apple juice processing. Molds were isolated at different steps and then identified by their macroscopic and microscopic characteristics after cultivation on standard media at 5, 25 and 37 °C, during 7 days. Among the 19 isolated found, 63% were identified as Penicillium with 50% belonging to the P. expansum specie. With regards to heat resistant molds, the species Neosartorya fischeri, Byssochlamys fulva and also the genus Eupenicillium sp., Talaromyces sp. and Eurotium sp. were isolated. The thermoacidophilic spore-forming bacteria were identified as A. acidoterrestris by a further investigation based on 16S rRNA sequence similarity. The large contamination found indicates the need for methods to eliminate or prevent the presence of these microorganisms in the processing plants in order to avoid both spoilage of apple juice and toxin production.

Survey of molds, yeast and Alicyclobacillus spp. from a concentrated apple juice productive process

Bacteria and molds may spoil and/or contaminate apple juice either by direct microbial action or indirectly by the uptake of metabolites as off-flavours and toxins. Some of these microorganisms and/or metabolites may remain in the food even after extensive procedures. This study aim to identify the presence of molds (including heat resistant species) and Alicyclobacillus spp., during concentrated apple juice processing. Molds were isolated at different steps and then identified by their macroscopic and microscopic characteristics after cultivation on standard media at 5, 25 and 37ºC, during 7 days. Among the 19 isolated found, 63% were identified as Penicillium with 50% belonging to the P. expansum specie. With regards to heat resistant molds, the species Neosartorya fischeri, Byssochlamys fulva and also the genus Eupenicillium sp., Talaromyces sp. and Eurotium sp. were isolated. The thermoacidophilic spore-forming bacteria were identified as A. acidoterrestris by a further investigation based on 16S rRNA sequence similarity. The large contamination found indicates the need for methods to eliminate or prevent the presence of these microorganisms in the processing plants in order to avoid both spoilage of apple juice and toxin production.

High-cell-density culture strategies for polyhydroxyalkanoate production: a review.

This article gives an overview of high-cell-density cultures for polyhydroxyalkanoate (PHA) production and their modes of operation for increasing productivity. High cell densities are very important in PHA production mainly because this polymer is an intracellular product accumulated in various microorganisms, so a high cellular content is needed for the polymer production. This review describes relevant results from fed-batch, repeated batch, and continuous modes of operation without and with cell recycle for the production of these polymers by microorganisms. Finally, recombinant microorganisms for PHA production, as well future directions for PHA production, are discussed.

Behaviour of Neosartorya fischeri Ascospores in pineapple juice

For heat-resistance derterminations, a Neosartorya fischeri ascospore suspension, obtained at three different conditions of temperature (20, 30 and 35ºC) and age (1, 2 and 3 months), was heat treated in pineapple juice with three different ratio conditions. The 1/k (min) values obtained were statistically analysed through the software Statisitca 6.0. At 80ºC, when there was a level change in the ratio factor from a lower (10) to a higher one (38), the thermal resistance (1/k) increased in approximately 41 per cent. The increase of the ascospore production age and temperature caused an increase on the thermal resistance approximately equal to 28 per cent and 14 per cent, respectively. Throufh the results, it could be concluded that N. fischeri presented lower thermal resistance for heating mediums with lower ratio values, or rather, the increase of the soluble solids concentration caused an increase of its thermal resistance. It was also observed that the ascospore production age and temperature increased significantly the thermal resistance of this mould in pineapple juice.

Avaliação da resistência térmica de Byssochlamys nivea E de Neosartorya fischeri em suco de abacaxi / Thermal resistance evaluation of Byssochllamys nivea E in pineapple juice

Estudou-se a resistência térmica de ascósporos dos fungos termorresistentes Byssochlamys nivea e Neosartorya fischeri, usando suco de abacaxi (pH 3,5 e 12ºBrix) como meio de aquecimento. Determinou-se a resistência térmica em Tempo de Destruição Térmica (TDT) nas temperaturas de 85,90 e 92 ºC. O maior valor de 1/k encontrado foi de 45 minutos para ascósporos de B. nivea a 85 ºC. Nessas mesmas condições, o valor de 1/k para N. fischeri foi de 30 minutos. Os resultados indicaram maior resist~encia térmica de ascósporos de B. nivea em comparação com ascósporos de N. fischeri em suco de abacaxi.

Factors affecting the thermal activation of Neosartorya fischeri in pineapple and papaya nectars

In this research factors that influence the activation of Neosartorya fischeri ascospores (heating medium, temperature of production and age of the ascospores) were studied. The heating medium used was pineapple and papaya nectars at different ratios (°Brix/acidity). Lower activation times were observed for the N. fischeri ascospores in pineapple and papaya nectars under the conditions of temperature and production age of 25°C for 1 month and using ratios of the heating medium of 10 for pineapple nectar and 26 for papaya nectar. In these cases, the activation time was 5 minutes for the pineapple nectar and 10 minutes for the papaya nectar at 85°C. For the ascospores produced at 35°C over three months, heated in pineapple nectar at a ratio of 38 and in papaya nectar at a ratio of 66, the longest activation times of 15 and 20 minutes, respectively, were observed. The statistical analysis showed that the factors which had the most influence in the activation time of the ascospores were the ratio and the production temperature of ascospores.

Nesta pesquisa, foram estudados fatores que influenciam a ativação de ascósporos do fungo termorresistente Neosartorya fischeri (meio de aquecimento, a temperatura de produção e idade dos ascósporos). Os meios de aquecimento utilizados foram sucos de abacaxi e de mamão, em diferentes ratios (°Brix/acidez) e a temperatura de ativação foi 85°C. Pôde-se observar que o fungo N. fischeri apresentou seus menores tempos de ativação quando presentes no suco de abacaxi e no mamão, nas condições de temperatura e de idade de produção de 25°C por 1 mês e o ratio do meio de aquecimento de 10 para suco de abacaxi e de 26 para suco de mamão. Nestes ensaios, o tempo de ativação foi de 5 minutos para suco de abacaxi e 10 minutos para o suco de mamão a 85°C. Ascósporos produzidos a 35°C, durante 3 meses e aquecidos em suco de abacaxi com ratio 38 e em suco de mamão com ratio 66 apresentaram os maiores tempos de ativação sendo estes de 15 a 20 minutos, respectivamente. Através de uma análise estatística, verificou-se que os fatores que mais influenciaram no tempo de ativação dos ascósporos foram o ratio e a temperatura de produção dos ascósporos.