Experimental and numerical study of heterogeneous pressure-temperature-induced lethal and sublethal injury of Lactococcus lactis in a medium scale high-pressure autoclave

The present contribution is dedicated to experimental and theoretical assessment of microbiological process heterogeneities of the high-pressure (HP) inactivation of Lactococcus lactis ssp. cremoris MG 1363. The inactivation kinetics are determined in dependence of pressure, process time, temperature and absence or presence of co-solutes in the buffer system namely 4 M sodium chloride and 1.5 M sucrose. The kinetic analysis is carried out in a 0.1-L autoclave in order to minimise thermal and convective effects. Upon these data, a deterministic inactivation model is formulated with the logistic equation. Its independent variables represent the counts of viable cells (viable but injured) and of the stress-resistant cells (viable and not injured). This model is then coupled to a thermo-fluiddynamical simulation method, high-pressure computer fluid dynamics technique (HP-CFD), which yields spatiotemporal temperature and flow fields occurring during the HP application inside any considered autoclave. Besides the thermo-fluiddynamic quantities, the coupled model predicts also the spatiotemporal distribution of both viable (VC) and stress-resistant cell counts (SRC). In order to assess the process non-uniformity of the microbial inactivation in a 3.3-L autoclave experimentally, microbial samples are placed at two distinct locations and are exposed to various process conditions. It can be shown with both, experimental and theoretical models that thermal heterogeneities induce process non-uniformities of more than one decimal power in the counts of the viable cells at the end of the treatment.     

The influence of transport phenomena during high-pressure processing of packed food on the uniformity of enzyme inactivation

Here we deal with the influence of heat-transport effects on a high-pressure-induced enzyme inactivation in packed substances. Special attention is given to the influence of the geometrical scale and to the heat-transfer characteristics of the packaging material. The investigation is based on mathematical modeling and numerical simulation. The method accounts both for compression phase and holding phase. The model includes convective and conductive heat transfer, fluid motion as well as an enzyme transport equation with a first-order kinetic source term accounting for the inactivation. Three configurations with a total volume of 0.8 L, 6.3 L, and 50.3 L are considered. The pressure medium is water. The enzyme solution is B. subtilis alpha-amylase dissolved in a TRIS-HCl-buffer. The packaging material is polypropylene. The heat-transfer coefficient for conduction through the packaging material is varied to simulate both changes in the material properties as well as modifications of the packaging material thickness. It is found that the efficiency of the inactivation increases with increasing chamber volume as long as the kinetic inactivation constant is increasing with temperature. In the considered case the activity retention obtained in a 0.8 L volume is about 2.4 times larger than the one obtained for the same process carried out in a 50.3 L volume. Furthermore, it was found that the properties of the packaging material could induce a significant degree of nonuniformity (worst case = 69%). An appropriate choice of the material can lead to maximum inactivation and maximum process uniformity since advantage is taken from the slow heat exchange after the compression phase.     

Purification, characterization, thermal, and high-pressure inactivation of pectin methylesterase from bananas (cv Cavendish)

Pectin methylesterase (PME) was extracted from bananas (cv Cavendish) and purified by affinity chromatography on a CNBr-Sepharose-PME inhibitor (PMEI) column. A single protein and PME activity peak was obtained. For banana PME, a biochemical characterization in terms of molar mass (MM), pI, and kinetic parameters was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal inactivation of purified banana PME could be described by a first-order kinetic model in a temperature range of 65 degrees to 72.5 degrees C, whereas its isobaric-isothermal inactivation followed a fractional-conversion model. Banana PME was found to be more thermally stable compared with PMEs extracted from orange, tomato, and apple.     

Numerical evaluation of lactoperoxidase inactivation during continuous pulsed electric field processing

A computational fluid dynamics (CFD) model describing the flow, electric field and temperature distribution of a laboratory‐scale pulsed electric field (PEF) treatment chamber with co‐field electrode configuration was developed. The predicted temperature increase was validated by means of integral temperature studies using thermocouples at the outlet of each flow cell for grape juice and salt solutions. Simulations of PEF treatments revealed intensity peaks of the electric field and laminar flow conditions in the treatment chamber causing local temperature hot spots near the chamber walls. Furthermore, thermal inactivation kinetics of lactoperoxidase (LPO) dissolved in simulated milk ultrafiltrate were determined with a glass capillary method at temperatures ranging from 65 to 80°C. Temperature dependence of first order inactivation rate constants was accurately described by the Arrhenius equation yielding an activation energy of 597.1 kJ mol^?1. The thermal impact of different PEF processes on LPO activity was estimated by coupling the derived Arrhenius model with the CFD model and the predicted enzyme inactivation was compared to experimental measurements. Results indicated that LPO inactivation during combined PEF/thermal treatments was largely due to thermal effects, but 5–12% enzyme inactivation may be related to other electro‐chemical effects occurring during PEF treatments. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Moderate temperatures affect Escherichia coli inactivation by high-pressure homogenization only through fluid viscosity

The inactivation of suspensions of Escherichia coli MG1655 by high-pressure homogenization was studied over a wide range of pressures (100-300 MPa) and initial temperatures of the samples (5-50 degrees C). Bacterial inactivation was positively correlated with the applied pressure and with the initial temperature. When samples were adjusted to different concentrations of poly(ethylene glycol) to have the same viscosity at different temperatures below 45 degrees C and then homogenized at these temperatures, no difference in inactivation was observed. These observations strongly suggest, for the first time, that the influence of temperature on bacterial inactivation by high-pressure homogenization is only through its effect on fluid viscosity. At initial temperatures > or =45 degrees C, corresponding to an outlet sample temperature >65 degrees C, the level of inactivation was higher than what would be predicted on the basis of the reduced viscosity at these temperatures, suggesting that under these conditions heat starts to contribute to cellular inactivation in addition to the mechanical effects that are predominant at lower temperatures. Second-order polynomial models were proposed to describe the impact of a high-pressure homogenization treatment of E. coli MG1655 as a function of pressure and temperature or as a function of pressure and viscosity. The pressure-viscosity inactivation model provided a better quality of fit of the experimental data and furthermore is more comprehensive and versatile than the pressure-temperature model because in addition to viscosity it implicitly incorporates temperature as a variable.     

Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: mechanism of protective action of alpha-crystallin

The kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscle have been studied by dynamic light scattering (0.08M Hepes, pH 6.8, containing 0.1M NaCl; 48 degrees C). The hydrodynamic radius of the start aggregates determined from the initial linear parts of the dependences of the hydrodynamic radius (R(h)) on time was found to be 16.7 +/- 1.0 nm. At rather high values of time, the R(h) value for the protein aggregates becomes proportional to t(1/1.8) = t(0.56) suggesting that the aggregation process proceeds in the regime of diffusion-limited cluster-cluster aggregation. In the presence of alpha-crystallin, a protein possessing the chaperone-like activity, the process of protein aggregation switches to the regime of reaction-limited cluster-cluster aggregation as indicated by the exponential dependence of the R(h) value on time. It was shown that the addition of alpha-crystallin raises the rate of thermal inactivation of Phb. These data in combination with the results of the study of interaction of Phb with alpha-crystallin by analytical ultracentrifugation suggest that alpha-crystallin interacts with the intermediates of unfolding of the Phb molecule.     

Fluid flow in a spiral device used for irradiation of biological fluids

The manufacture of plasma‐derived therapeutics includes dedicated viral inactivation steps to minimize the risk of infection. Traditional viral inactivation methods are effective for the removal and inactivation of enveloped viruses, but less effective against small nonenveloped viruses. UV‐C irradiation has been demonstrated to be an effective means of inactivating such viruses. The UVivatec lab system consists of a spiral tube around an UV‐C irradiation source. Flow of a solution through the chamber generates and ensures controlled mixing and uniform exposure to irradiation. A detailed assessment of the effect of flow rate, alternate cross sectional design and scale up of the irradiation chamber on Dean vortices was performed using the smoothed particle hydrodynamics method. The aim was to provide a basis for setting flow rate limits and using a laboratory scale apparatus to model viral inactivation in larger manufacturing scale equipment. The effect of flow rate related changes on the fluence rate was also investigated through chemical actinometry studies. The data were consistent with the simulations indicating that Dean vortices were present at low flow rates, but dissipated at higher flow rates through the spiral chamber. Importantly, this work also allowed a correlation between the small system and large scale system to be established. This will greatly facilitate process development and viral validation studies. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 359–367, 2013     

Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery

Campylobacter jejuni is one of the most intriguing human foodborne bacterial pathogen. Its survival throughout the food processing chain and its pathogenesis mechanisms in humans remain enigmatic. Living in the animal guts and particularly in avian intestine as a commensal bacterium, this microorganism is frequently isolated from meat products. Ultra high pressure (HP) is a promising alternative to thermal technology for microbial safety of foodstuffs with less organoleptic and nutritional alterations. Its application could be extended to meat products potentially contaminated by C. jejuni. To evaluate the response of Campylobacter to this technological stress and subsequent recovery at a molecular level, a dynamic 2-DE-based proteomic approach has been implemented. After cultivation, C. jejuni cells were conditioned in a high-pressure chamber and transferred to fresh medium for recovery. The protein abundance dynamics at the proteome scale were analyzed by 2-DE during the cellular process of cell injury and recovery. Monitoring protein abundance through time unraveled the basic metabolisms involved in this cellular process. The significance of the proteome evolution modulated by HP and subsequent recovery is discussed in the context of a specific cellular response to stress and recovery of C. jejuni with 69 spots showing significant changes through time.     

High-pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: refolding optimization, and feasibility assessment

High-pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1-3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large-scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29-42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high-pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high-pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale-up for this molecule.     

Muscle contraction: viscous-like frictional forces and the impulsive model

Apart from a few experimental studies muscle viscosity has not received much recent analytical attention as a determinant of the contractile process. This is surprising, since any muscle cell is 80% water, and may undergo large shape changes during its working cycle. Intuitively one might expect the viscosity of the solvent to be an important determinant of the physiological activity of muscle tissue. This was apparent to pioneers of the study of muscle contraction such as Hill and his contemporaries, whose putative theoretical formulations contained terms related to muscle viscosity. More recently, though, a hydrodynamic calculation by Huxley, using a solvent viscosity close to that of water, has been held to demonstrate that viscous forces are negligible in muscle contraction. We have re-examined the role of viscosity in contraction, postulating impulsive acto-myosin forces that are opposed by a viscous resistance between the filaments. The viscous force required, 10⁴ times the hydrodynamic estimate, is close to recent experimental measurements, themselves 10²–10³ times the hydrodynamic estimate. This also agrees with contemporary measurements of cytoplasmic viscosity in other biological cells using magnetic bead micro-rheometry. These are several orders of magnitude greater than the viscosity of water. In the course of the analysis we have derived the force-velocity equation for an isolated half-sarcomere containing a single actin filament for the first time, and from first principles. We conclude that muscle viscosity is indeed important for the contractile process, and that it has been too readily discounted.     

A method to rationally increase protein stability based on the charge–charge interaction,with application to lipase LipK107

We report a suite of enzyme redesign protocol based on the surface charge–charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with ～10°C higher half‐inactivation temperature than wild‐type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14‐fold increase in the half‐inactivation time at 50°C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.     

Thermal inactivation of D-amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a long-known flavoenzyme whose most important biocatalytic application is currently the industrial production of 7-amino-cephalosporanic acid (7-ACA) from cephalosporin C. Lacking mechanistic foundation, rational stabilization of TvDAO for improved process performance remains a problem. We report on results of thermal denaturation studies at 50 degrees C in which two purified TvDAO forms were compared: the native enzyme, and a site-specifically oxidized protein variant that had the side chain of cysteine108 converted into a sulfinic acid and lost 75% of original specific activity. Although inactivation time courses for both enzymes are fairly well described by simple single-exponential decays, the underlying denaturation mechanisms are shown by experiments and modeling to be complex. One main path leading to inactivation is FAD release, a process whose net rate is determined by the reverse association rate constant (k), which is 25-fold lower in the oxidized form of TvDAO. Cofactor dissociation is kinetically coupled to aggregation and can be blocked completely by the addition of free FAD. Aggregation is markedly attenuated in the less stable Cys108-SO(2)H-containing enzyme, suggesting that it is a step accompanying but not causing the inactivation. A second parallel path, characterized by a k-value of 0.26/h that is not dependent on protein concentration and identical for both enzymes, likely reflects thermal unfolding reactions. A third, however, slow process is the conversion of the native enzyme into the oxidized form (k < 0.03/h). The results fully explain the different stabilities of native and oxidized TvDAO and provide an inactivation mechanism-based tool for the stabilization of the soluble oxidase.     

Two-dimensional plasma density distributions in low-pressure gas discharges

The plasma density distribution in a two-dimensional nonuniform positive column of a low-pressure gas discharge is studied in the hydrodynamic approximation with allowance for ion inertia. Exact solutions are derived for discharges in a rectangular and a cylindrical chamber. Asymptotic solutions near the coordinate origin and near the critical surface are considered. It is shown that, for potential plasma flows, the flow velocity component normal to the plasma boundary is equal to the ion acoustic velocity. The results obtained can be used to analyze the processes occurring in low-pressure plasmochemical reactors.     

Effect of pH in the acidic region on the structural integrity of lipase from wheat germ

To understand the structure-function relationship of the enzyme lipase the effect of acid pH on the activity of lipase has been followed using a number of physico-chemical techniques. Lipase from wheat/germ has S20,w value of 2.2 S and a molecular weight of 42,000 +/- 1,000. The enzyme has an intrinsic viscosity of 4.72 ml/g indicating it to be elongated in shape. With decrease in pH below 7.0 microenvironmental changes occur in the neighborhood of active site accompanied by minor conformational changes without any gross change in the hydrodynamic properties of the protein, as monitored with ultraviolet difference spectra, fluorescence spectra, viscosity and circular dichroism. The kinetics of the inactivation process has been established as consisting of a fast step and a slow step with a k value of 73/sec and 7.2/sec respectively. At extreme acid pH the enzyme reaggregates to a polymer arising out of hydrophobic interaction and the polymer has no activity.     

Derivation of radiation quality average parameters in neutron-gamma radiation fields with the high-pressure ionization chamber: theory and practice

The established radiation quality parameters in mixed neutron-gamma radiation fields may be measured by applying the initial (columnar) recombination of ions in tissue-equivalent (TE) high-pressure ionization chambers (recombination chambers). The mean quality factor can be determined to within 10-15% for mixed fields with neutrons ranging from thermal to 10 MeV, and the dose mean LET of the proton component can be determined to within 10-15% if the gamma-ray absorbed dose fraction is known. These average parameters are derived by measuring the ratio of the ionization currents collected at two high-field strengths and constant gas pressure applied to the ionization chamber. By utilizing approximate correlations between physical parameters in the neutron energy region from thermal to 10 MeV, the dose mean LET of the heavy ion component, the overall dose mean LET, and the microdosimetric parameter y0,D of the mixed field can also be derived. Experimental verification of the method is presented for various neutron-gamma radiation spectra in air and in water by comparison to theoretical calculations and results from low-pressure proportional counter measurements. Good agreement is shown. The TE high-pressure ionization chamber appears to have wide potential for use as a dose-equivalent meter in radiation protection or as a beam characterization device in radiobiology.     

Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.     

Strawberry pectin methylesterase (PME): purification,characterization, thermal and high-pressure inactivation

Pectin methylesterase (PME) was extracted from strawberries (Fragaria ananassa, cv Elsanta) and purified by affinity chromatography on a CNBr-Sepharose 4B-PME-inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molecular mass, pI, and kinetic parameters of strawberry PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified strawberry PME could be described by a fractional-conversion model. Purified strawberry PME is much more stable toward high-pressure treatments in comparison to those from oranges and bananas.     

A simple approximation for larval retention around reefs

Estimating larval retention at individual reefs by local scale three-dimensional flows is a significant problem for understanding, and predicting, larval dispersal. Determining larval dispersal commonly involves the use of computationally demanding and expensively calibrated/validated hydrodynamic models that resolve reef wake eddies. This study models variation in larval retention times for a range of reef shapes and circulation regimes, using a reef-scale three-dimensional hydrodynamic model. It also explores how well larval retention time can be estimated based on the “Island Wake Parameter”, a measure of the degree of flow turbulence in the wake of reefs that is a simple function of flow speed, reef dimension, and vertical diffusion. The mean residence times found in the present study (0.48–5.64 days) indicate substantial potential for self-recruitment of species whose larvae are passive, or weak swimmers, for the first several days after release. Results also reveal strong and significant relationships between the Island Wake Parameter and mean residence time, explaining 81–92% of the variability in retention among reefs across a range of unidirectional flow speeds and tidal regimes. These findings suggest that good estimates of larval retention may be obtained from relatively coarse-scale characteristics of the flow, and basic features of reef geomorphology. Such approximations may be a valuable tool for modeling connectivity and meta-population dynamics over large spatial scales, where explicitly characterizing fine-scale flows around reef requires a prohibitive amount of computation and extensive model calibration.     

Modeling of scale-down effects on the hydrodynamics of expanded bed adsorption columns

Expanded-bed adsorption (EBA) is a technique for primary recovery of proteins starting from unclarified broths. This process combines centrifugation, concentration, filtration, and initial capturing of the proteins in a single step. An expanded bed (EB) is comparable to a packed bed in terms of separation performance but its hydrodynamics are that of a fluidized bed. Downstream process development involving EBA is normally carried out in small columns to minimize time and costs. Our purpose here is to characterize the hydrodynamics of expanded beds of different diameters, to develop scaling parameters that can be reliably used to predict separation efficiency of larger EBA columns. A hydrodynamic model has been developed which takes into account the radial liquid velocity profile in the column. The scale-down effect can be characterized in terms of apparent axial dispersion, D(axl,app), and plate number, N(EB), adapted for expanded bed. The model is in good agreement with experimental results obtained from 1- and 5-cm column diameters with buffer solutions of different viscosities. The model and the experiments show an increase of apparent axial dispersion with an increase in column diameter. Furthermore, the apparent axial dispersion is affected by an increase in liquid velocity and viscosity. Supported by visual observations and predictions from the model, it was concluded that operating conditions (liquid viscosity and superficial velocity) resulting in a bed-void fraction between 0.7 and 0.75 would provide the optimal separation efficiency in terms of N(EB).     

Leveraging flow mechanics to determine critical process and scaling parameters in a continuous viral inactivation reactor

A continuous viral inactivation (CVI) chamber has been designed to operate with acceptable residence time distribution (RTD) characteristics. However, altering the CVI's geometry and operation to accommodate the scale was not obvious. In this work, we elucidate the influence of Dean vortices and leverage the transition into the weak turbulent regime to establish relationships between input variables and process outputs. This study was targeted to understand and quantify the impact of viscosity, Dean number, internal diameter, and path length on the RTD. When the Dean number exceeds 70, radial mixing generated by the Dean vortices began to consistently alter the axial dispersive effects experienced by the pulse injection. Increasing to a Dean number of >100, the axial dispersive effects were dominated by the Dean vortices which allowed the calculation of the minimum and maximum residence time to be generated. This work provides a method to calculate operational solutions for a tubular incubation reactor in terms of path length, internal diameter, flow rate, and target minimum and maximum residence time specifications that assures both viral residence times while also establishing criteria to maximize product quality during continuous operation.