Relevance of microbial interactions to predictive microbiology.

Microbial interaction can be ignored in predictive microbiology under most conditions. We show that interactions are only important at high population densities, using published data on inhibition of growth of Listeria monocytogenes in broth. Our analysis using growth models from predictive microbiology indicated that interactions only occur at population densities of approximately 10(8) cfu/ml of the protective cultures. Spoilage is evident at these levels, except for fermented foods. In bacterial colonies, diffusion limitation acts as a constraint to growth. We have shown that these constraints only become important after large outgrowth of colonies (in the order of 5-log growth in Lactobacillus curvatus colonies), which depends on the initial inoculation density. Intra-colony interactions play an important role under these conditions. There is no large outgrowth of colonies when the initial inoculation densities are high and broth culture growth can be used to approximate colony growth.

Modeling the interactions of Lactobacillus curvatus colonies in solid medium: consequences for food quality and safety.

The growth process of Lactobacillus curvatus colonies was quantified by a coupled growth and diffusion equation incorporating a volumetric rate of lactic acid production. Analytical solutions were compared to numerical ones, and both were able to predict the onset of interaction well. The derived analytical solution modeled the lactic acid concentration profile as a function of the diffusion coefficient, colony radius, and volumetric production rate. Interaction was assumed to occur when the volume-averaged specific growth rate of the cells in a colony was 90% of the initial maximum rate. Growth of L. curvatus in solid medium is dependent on the number of cells in a colony. In colonies with populations of fewer than 10(5) cells, mass transfer limitation is not significant for the growth process. When the initial inoculation density is relatively high, colonies are not able to grow to these sizes and growth approaches that of broth cultures (negligible mass transfer limitation). In foods, which resemble the model solid system and in which the initial inoculation density is high, it will be appropriate to use predictive models of broth cultures to estimate growth. For a very low initial inoculation density, large colonies can develop that will start to deviate from growth in broth cultures, but only after large outgrowth.

The effect of ethanol on the kinetics of lipase-mediated enantioselective esterification of 4-methyloctanoic acid and the hydrolysis of its ethyl ester.

The Novozym 435(R) catalyzed esterification and hydrolysis reactions of 4-methyloctanoic acid (ethyl ester) were investigated. In both the hydrolysis and esterification reactions, the increase of ethanol concentration led to an increase in enantiomeric ratio (E). For hydrolysis of the ethyl ester, the E-value increased from 5.5 [0% (v/v) EtOH] up to 12 [20% (v/v) EtOH]. In case of esterification, the E-value was already 16 [14% (v/v) EtOH] and rose to 57 [73% (v/v) EtOH]. When combining these results of esterification and hydrolysis, an enantiomeric ratio of 350 can be estimated for the sequential kinetic resolution of 4-methyloctanoic acid. In this way, enantiopure 4-methyloctanoic acid could be obtained after two consecutive reaction steps.

Enzymatic synthesis of oligosaccharides: product removal during a kinetically controlled reaction.

In this article, the enzymatic production of oligosaccharides, which is an example of a kinetically controlled reaction, is studied. The aim is to show that the product yield can be enhanced by continuous removal of oligosaccharides from the reaction mixture. The oligosaccharides were removed by adsorption on activated carbon. The absorption could be described by the multicomponent Langmuir isotherm with different maximum saturation constants for mono-, di-, and trisaccharides. The affinity for trisaccharides was larger (k(tri) = 3.52 l/g) than for di- (k(di) = 0.94 l/g) and monosaccharides (k(mono) = 0.11 l/g). A model combining kinetics, adsorption on activated carbon, and mass transfer in an adsorption column was developed. Model calculations for the batch process with removal showed a yield improvement of 23% compared to the batch process without removal. Experimentally, a yield improvement of 30% was obtained. Model calculations for the continuous process studied did not result in an increase of the yield. The advantages of removal were masked by the negative influence of recirculation and the relative large time between formation and removal.

Microgradients in bacterial colonies: use of fluorescence ratio imaging, a non-invasive technique.

Fluorescence ratio imaging is a non-invasive technique for studying the formation of microgradients in immobilised bacterial colonies. These gradients can be quantified easily when combined with the gel cassette system designed at the Institute of Food Research, Norwich, UK. Colonies of Lactobacillus curvatus were observed using this technique and relevant pH gradients were present when the colonies reached a diameter of about 100 microm. These pH gradients were due to production of lactic acid by L. curvatus cells in the colonies. The spatial resolution of the images was about 1.5 microm (scale of bacterial cells) and therefore very suitable for observing local effects in colonies which ranged in sizes from 1 to 500 microm.

Stepwise quantitative risk assessment as a tool for characterization of microbiological food safety.

This paper describes a system for the microbiological quantitative risk assessment for food products and their production processes. The system applies a stepwise risk assessment, allowing the main problems to be addressed before focusing on less important problems. First, risks are assessed broadly, using order of magnitude estimates. Characteristic numbers are used to quantitatively characterize microbial behaviour during the production process. These numbers help to highlight the major risk-determining phenomena, and to find negligible aspects. Second, the risk-determining phenomena are studied in more detail. Both general and/or specific models can be used for this and varying situations can be simulated to quantitatively describe the risk-determining phenomena. Third, even more detailed studies can be performed where necessary, for instance by using stochastic variables. The system for quantitative risk assessment has been implemented as a decision supporting expert system called SIEFE: Stepwise and Interactive Evaluation of Food safety by an Expert System. SIEFE performs bacterial risk assessments in a structured manner, using various information sources. Because all steps are transparent, every step can easily be scrutinized. In the current study the effectiveness of SIEFE is shown for a cheese spread. With this product, quantitative data concerning the major risk-determining factors were not completely available to carry out a full detailed assessment. However, this did not necessarily hamper adequate risk estimation. Using ranges of values instead helped identifying the quantitatively most important parameters and the magnitude of their impact. This example shows that SIEFE provides quantitative insights into production processes and their risk-determining factors to both risk assessors and decision makers, and highlights critical gaps in knowledge.

Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides.

The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters. A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.

Modelling the interactions between Lactobacillus curvatus and Enterobacter cloacae. I. Individual growth kinetics.

Mixed cultures of Lactobacillus curvatus and Enterobacter cloacae were chosen as a model system to quantitatively study microbial interactions involved in food spoilage and food preservation. In this paper models were developed to predict the individual behaviour of L. curvatus and E. cloacae in pure suspension cultures as a function of the glucose and lactate concentration and the pH. In a second paper these models will be used to predict the moment of interaction in mixed suspension cultures and the cell concentrations at this moment. The effect of pH on the maximum specific growth rate could be described by a parabolic equation for L. curvatus and E. cloacae. E. cloacae is clearly more sensitive to a pH decrease than L. curvatus, as may be concluded from the theoretical minimum pH for growth of 5.6 for E. cloacae and 4.3 for L. curvatus. For both organisms no effect of glucose on the maximum specific growth rate could be detected and Monod kinetics with a low Monod constant was assumed. For L. curvatus the effect of lactate on the maximum specific growth rate at different initial pH values could be described by a linear relationship. For E. cloacae a slight effect of lactate on the maximum specific growth rate could only be detected at pH 7. However, this effect was negligible compared to the effect of the pH. For both organisms the lag time was modelled by the inverse of the specific growth rate. It can be concluded that, with regard to interactions between L. curvatus and E. cloacae, the pH is likely to be the most important factor in the case where L. curvatus is the dominant organism. Substrate limitation is likely to become important in the case where E. cloacae is the dominant organism or for media with a high buffer capacity.

Modelling the interactions between Lactobacillus curvatus and Enterobacter cloacae. II. Mixed cultures and shelf life predictions.

The modelling approach presented in this study can be used to predict when interactions between microorganisms in homogenous systems occur. It was tested for the interaction between Lactobacillus curvatus and Enterobacter cloacae. In this binary system, L. curvatus produces lactic acid which decreases the pH in the system. The pH decrease was found to be the main limiting factor of growth of both E. cloacae and L. curvatus. This resulted in E. cloacae reaching its final concentration earlier when compared to its growth in pure culture. The models consisted of a set of first order ordinary differential equations describing the growth, consumption and production rates of both microorganisms. The parameters for these equations were obtained from pure culture studies and from literature. These equations were solved using a combination of analytical and numerical methods. The prediction of growth in mixed culture using parameters from pure culture experiments and literature were close to the experimental data. Both model predictions and experimental validation indicated that interaction occurs when the concentration of L. curvatus reaches 10(8) cfu/ml. At that moment in time, the pH had decreased to inhibiting levels. These concentrations of microorganisms (10(8) cfu/ml) do occur in fermented products where interactions obviously are important. In nonfermented foods however, this level of microorganisms indicate that spoilage has occurred or is about to start. Microbial interactions can therefore be neglected when predicting shelf life or safety of food products in most cases.

A data analysis of the irradiation parameter D10 for bacteria and spores under various conditions.

This paper provides approximate estimates for the irradiation parameter D10 to globally predict the effectiveness of any irradiation process. D10 is often reported to depend on many specific factors, implying that D10 cannot be estimated without exact knowledge of all factors involved. For specific questions these data can of course be useful but only if the conditions reported exactly match the specific question. Alternatively, this study determined the most relevant factors influencing D10, by quantitatively analyzing data from many references. The best first step appeared to be a classification of the data into vegetative bacteria and spores. As expected, spores were found to have significantly higher D10 values (average 2.48 kGy) than vegetative bacteria (average 0.762 kGy). Further analyses of the vegetative bacteria confirmed the expected extreme irradiation resistance of nonpathogenic Deinococcus radiodurans (average 10.4 kGy). Furthermore the analysis identified Enterococcus faecium, Alcaligenes spp., and several members of the Moraxella-Acinetobacter group as having very high resistance at very low temperatures (average 3.65 kGy). After exclusion of high- and low-resistance spores and some specific conditions showing relevant high or low D10 values, the average for spores was estimated to be 2.11 kGy. For vegetative bacteria this average was estimated to be 0.420 kGy. These approximate estimates are not definite, as they depend on the data used in the analyses. It is expected that inclusion of more data will not change the estimates to a great extent. The approximate estimates are therefore useful tools in designing and evaluating irradiation processes.

A decision support system for the prediction of microbial food safety and food quality.

The development of a method to predict microbial food safety and quality is described. The manufacture of a food from its ingredients is simulated, using a recipe. Food engineering heuristics are combined with models developed in predictive microbiology. Parameter values of ingredients of foods, such as water activity and pH, and models for microbial growth and decay are used for the prediction of the kinetics of microorganisms generally found in ingredients. The values of these parameters are collected in databases. If required information is lacking, methods are described for making reliable guesses of the parameters. Food quality can be calculated as a function of fluctuating temperature in time. Several food distribution chains can be simulated in order to assess the influence of distribution chains on food quality. The described methods were implemented into a computerised decision support system that can be used in food production, product development and training. In the future it may be possible to apply specific expert knowledge in production and development of foods to improve the quality of prediction.

Combined influence of growth and drying conditions on the activity of dried lactobacillus plantarum

The production of active dried starter cultures can be influenced at several levels in the production process. In this paper the following process factors are discussed: osmotic stress during growth and cell density prior to drying. Contradicting results are reported in the literature on the influence of osmotic stress during growth on the residual activity after drying. The combined approach in which two process factors were studied at a time resulted in an explanation for the discrepancy in earlier work. The cell density prior to drying had an important influence on the glucose fermenting activity after drying. Residual activities ranging from 0.10 to 0.83 were achieved using initial cell densities between 0.025 and 0.23 g of cell/g of sample, respectively. The drying tolerance of cells grown with osmotic stress of 1 M NaCl was low (residual activity = 0. 06) and was not related to the cell density prior to drying. The influence of osmotic stress during growth on the drying tolerance of Lactobacillus plantarum was dependent on the cell density prior to drying.

Inactivation Kinetics Study of the Kunitz Soybean Trypsin Inhibitor and the Bowman-Birk Inhibitor.

The inactivation of trypsin inhibitors (TIs) in soy flour exhibits a two-phase inactivation behavior. It is sometimes assumed that this behavior is caused by a difference in the heat stabilities of the Kunitz soybean trypsin inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). Kinetics studies with KSTI and BBI in soy flour showed that this two-phase inactivation behavior of TIs could not be explained by the difference in the heat stabilities of KSTI and BBI. Inactivation of KSTI and BBI in an aqueous solution and in a starch matrix followed a first-order reaction. KSTI and BBI in a starch matrix with added cysteine showed a two-phase inactivation behavior. The existence of thiols in soy flour seems to be responsible for the two-phase inactivation of TIs in soy flour. It is suggested that TIs in soy flour are inactivated by sulfhydryl-disulfide interchange during the first inactivation phase and by heat during the second phase.

Effect of Blanching on Structural Quality of Dried Potato Slices.

Mechanical properties of potato slices were monitored during blanching, as indicators of structural changes. As expected, blanching resulted in weakening of potato structure. Gelatinization, which occurred during the first 2 min, did not promote an immediate weakening of the potato tissue. More than 80% of the changes in mechanical properties occurred during the first 30 min of blanching. Potato slices blanched for 2 and 30 min as well as unblanched ones were dried in a convective air drier at 48 degrees C. Bulk and true density, porosity, and shrinkage were monitored with time. Blanched potatoes resulted in a significantly more compact, less porous product with lower effective water diffusivity than unblanched potatoes. The results indicated that changes that occurred during the first 2 min of blanching had a much greater influence on structural quality of dried potatoes than changes that occurred from 2 to 30 min of blanching.

Effect of added carbohydrates on membrane phase behavior and survival of dried Lactobacillus plantarum.

The relation between the protective effect of externally added carbohydrates on Lactobacillus plantarum cells during air-drying and the phase behavior of cell membranes was studied. The residual activity after drying could be improved from 44% in the control to 79 and 66% after the addition of sorbitol and maltose, respectively, whereas trehalose addition resulted in a residual activity of 30%. Membrane phase transition temperatures (Tm) were determined in intact hydrated and dry cells, using Fourier transform infrared spectroscopy. The Tm of hydrated cells was 4 degrees C, increasing to only 20 degrees C after drying. Because endogenous soluble sugars were absent, this phase behavior is attributed to the structure of the predominant phospholipids, PG and lysyl-PG. The restricted increase of Tm is held responsible for the survival of part of the cells. The added maltose, trehalose, and sorbitol did not influence Tm in vivo. We suggest that the effective carbohydrates act through their free radical scavenging activity and not by direct interaction with the polar lipid headgroups.

Separation of amino-acid enantiomers using micellar-enhanced ultrafiltration.

Micellar-enhanced ultrafiltration (MEUF) is investigated as a large-scale technique for separating amino acid enantiomers. Specifically, L-5-cholesterol glutamate, a chiral ligand-exchange cosurfactant, is used together with a nonionic surfactant to form mixed micelles that preferentially bind D-phenylalanine over L-phenylalanine in the presence of copper(II). Operational selectivities as high as 4.2 are obtained. Potentiometric titrations using a water-soluble model compound similar to the chiral cosurfactant indicate that the ternary copper complex with phenylalanine has a stereoselectivity for the D enantiomer which is significantly smaller than that observed in the MEUF system. Thus, the selectivity of the chiral legend's local solvent and structural environment.

Evaluation of data transformations and validation of a model for the effect of temperature on bacterial growth.

The temperature of chilled foods is an important variable for controlling microbial growth in a production and distribution chain. Therefore, it is essential to model growth as a function of temperature in order to predict the number of organisms as a function of temperature and time. This article deals with the correct variance-stabilizing transformation of the growth parameters A (asymptotic level), mu (specific growth rate), and lambda (lag time). This is of importance for the regression analysis of the data. A previously gathered data set and model for the effect of temperature on the growth of Lactobacillus plantarum (M. H. Zwietering, J. T. de Koos, B. E. Hasenack, J. C. de Wit, and K. van 't Riet, Appl. Environ. Microbiol. 57:1094-1101, 1991) is extended with new data. With the total data set (original and new data), a variance-stabilizing transformation is selected in order to determine which transformation should precede fitting. No transformation for the asymptote data, a square root for the growth rate, and a logarithmic transformation for the lag time were found to be appropriate. After these transformations, no significant correlation was found between the variance and the magnitude of the variable. Model corrections were made and model parameters were estimated by using the original data. With the new data, the models were validated by comparing the lack of fit of the models with the measurement error, using an F test. The predictions of the models for mu and lambda were adequate. The model for A showed a systematic deviation, and therefore a new model for A is proposed.

Modeling of bacterial growth with shifts in temperature.

The temperature of chilled foods is an important variable for the shelf life of a product in a production and distribution chain. To predict the number of organisms as a function of temperature and time, it is essential to model the growth as a function of temperature. The temperature is often not constant in various stages of distribution. The objective of this research was to determine the effect of shifts in temperature. The suitability and usefulness of several models to describe the growth of Lactobacillus plantarum with fluctuating temperatures was evaluated. It can be assumed that temperature shifts within the lag phase can be handled by adding relative parts of the lag time to be completed and that temperature shifts within the exponential phase result in no lag phase. With these assumptions, the kinetic behavior of temperature shift experiments was reasonably well predicted, and this hypothesis was accepted statistically in 73% of the cases. Only shifts of temperature around the minimum temperature for growth showed very large deviations from the model prediction. The best results were obtained with the assumption that a temperature shift (within the lag phase as well as within the exponential phase) results in an additional lag phase. This hypothesis was accepted statistically in 93% of the cases. The length of the additional lag phase is one-fourth of the lag time normally found at the temperature after the shift.

Convective drying of bacteria. II. Factors influencing survival.

In the previous part of this review, the parameters of the drying process that can be important for the survival of bacteria upon drying, were reviewed. In this part the other factors which can be important for survival, will be discussed. The discussion starts with the mechanisms that can be responsible for thermal and dehydration inactivation. Moreover, the influence of storage conditions on the stability of dried bacterial cultures will be reviewed.