F.M. Rombouts

Development and validation of a combined temperature, water activity, pH model for bacterial growth rate of Lactobacillus curvatus

A model was established to predict growth rate as a function of temperature, pH and water activity. The model is based on two, earlier developed models, one for growth rate as a function of temperature and water activity and the other for growth rate as a function of temperature and pH. Based on the assumption that combinatory effects between pH and water activity do not exist, the two models were multiplied to produce one overall model. The overall model was then fitted to data sets measured earlier, and the parameters of the model were determined. A new data set with values for controlling variables outside the range of the earlier developed model was then used to validate the overall model statistically. The model was well able to extrapolate outside the measured data range. Finally, the model was updated with all measured data. No significant changes in the parameters were found. The approach followed underpins the gamma concept, since in the gamma concept it is assumed that the effects of controlling variables can be multiplied, and cardinal parameters are not a function of other variables (temperature, pH, and water activity).

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.

Crosslinked potato starch as an affinity adsorbent for bacterial α-amylase

Abstract Crosslinked potato starch was prepared as an affinity adsorbent for bacterial α-amylase. To this end, reaction parameters for crosslinking in an ethanol/water solvent were investigated. The degree of crosslinking, and consequently the suitability of crosslinked starch as an adsorbent for α-amylase, changed by altering these parameters. An increase in the degree of crosslinking of the adsorbent caused lower affinity for bacterial α-amylase which resulted in an unfavourable decrease in adsorption capacity and a favourable decrease in the degradation of the adsorbent by the enzyme. 1 g of a suitable adsorbent for bacterial α-amylase, prepared with an epichlorohydrin/glucose monomer ratio of 0·65 (starch concentration 150 mg/ml, ethanol/water ratio 2·0, sodium hydroxide/epichlorohydrin ratio 1·0), can adsorb 9·8 mg of an α-amylase from B. licheniformis at 4°C in 20 h. The equilibrium constant between bound and unbound α-amylase is dependent on the temperature. An effective desorption was possible by a shift to higher temperatures. Degradation values smaller than 0·1% were measured after an incubation of 1 h at 70°C in a desorption buffer with 20% glycerol. It was concluded that coulombic interactions and hydrogen bonds are of no or little importance in enzyme adsorption. Van der Waals forces, which are responsible for the large temperature effect, are the main forces in the interaction between α-amylase and crosslinked starch.

Developments in downstream processing of (poly)saccharide converting enzymes.

Recently novel techniques have been developed, based on liquid-liquid extractions and affinity interactions. Although they show considerable potential for the purification of several industrial enzymes they have not been widely introduced into existing processes yet. The introduction and applications of these techniques in the field of polysaccharidases are discussed

Some aspects of modelling microbial quality of food.

The kinetics of food deterioration reactions are important for the optimization of food chains. Different model types used to predict deterioration kinetics are briefly commented on. Objectives and needs for modelling are presented. A procedure to compare different models is given together with an example of their use. Some possibilities for expanding modelling techniques into decision support systems are given. Predictive models, kinetic data, expertise, logistics and simulation and optimization routines can be combined to support decisions in production and distribution and product development.

On the interaction of α-amylase with crosslinked starch: Evaluation of process conditions

Abstract The interaction of α-amylase with crosslinked starch is described. The adsorption characteristics are influenced especially by pH and temperature. Adsorption preferentially takes place at 4°C. The adsorption behavior corresponds with the catalytic activity of the enzymes studied. α-Amylase of Bacillus licheniformis , which has a broad pH optimum, adsorbs over a larger range (pH 5.0–9.0) than the α-amylase from Bacillus subtilis (pH 5.0–7.0). Capacities and Langmuir constants were determined in the relevant pH range. At pH values of 9.0–11.0 the catalytic activity and the adsorption levels drop, but the enzyme activity is not irreversibly lost. These conditions are used to recover the enzyme from the matrix. The crosslinked starch matrix is a competitive inhibitor for the enzyme in the enzyme assay. The K i was determined to be 6–8 g ml −1 for the inhibition of B. licheniformis α-amylase. The affinity for soluble starch appears to be approximately 30 times higher than for the matrix. As a result, limit dextrin solutions can be used as competitive eluents for the recovery of the enzyme from the adsorbent. A temperature shift from 4°C to 70°C can be used to recover the enzyme from the adsorbent, although this makes the matrix susceptible to biodegradation and enzyme activity is lost. The latter effect can be reduced by adding Ca 2+ to the system. Sodium chloride and glycerol have an influence on the interaction between α-amylase and the adsorbent. V max of the enzyme and the adsorption levels of α-amylase decrease among others as the water activity of the system is lowered. The matrix adsorbs a variety of α-amylases from bacterial and mammalian origin.

Crosslinked xylan as an affinity adsorbent for endo-xylanases

Abstract In order to facilitate the purification of xylanases from Aspergillus niger, an affinity adsorbent has been developed from oat spelts xylan. A suitable adsorbent was only obtained by crosslinking oat spelts xylan with epichlorohydrin in water but not in ethanol or ethanol-water mixtures. After some initial degradation of the adsorbent (approximately 4%), no significant biodegradation was measured with a reused adsorbent. Up to 60% of the xylanase activity from an Aspergillus niger enzyme mixture (50 mU/ml) was adsorbed at pH 4 (50 m m sodium acetate buffer). The degree of adsorption to crosslinked xylan of four fractions of this preparation, previously separated by DEAE-Biogel A chromatography, varied between 40 and 90%. Adsorption was strongly dependent on pH and ionic strength and desorption was easily accomplished by an increase in ionic strength. In addition to xylanases, polygalacturonases were also adsorbed to the matrix. No significant adsorption of β- d -xylosidase, α- l -arabinofuranosidase, β- d -galactosidase, β-(1,4)-galactanase, β-(1- 3 6 )- d -galactanase or cellulase activities was found.