Trehalase activity in extracts of Phycomyces blakesleeanus spores following the induction of germination by heat activation

The heat activation of trehalase in extracts of sporangiospores of Phycomyces blakesleeanus, following the induction of germination by heat activation and the gelatinization of potato starch granules were studied under different conditions in order to discriminate between several phenomena as possible triggers in the activation of trehalase.Short-chain alcohols (from methanol to pentanol) lower the activation temperature of trehalase while long-chain alcohols (from heptanol to nonanol) raise it. Short-chain alcohols also lower the gelatinization temperature of potato starch granules, while long-chain alcohols, hexanol and heptanol have hardly any influence on the gelatinization temperature. Octanol raises the gelatinization temperature. More polar phenols lower the activation temperature of trehalase, while more apolar phenols will raise it. The gelatinization temperature of starch granules is more lowered by the polar polyphenols than by the more apolar phenols.The effect of high pressure on starch gelatinization was investigated in order to compare data from such a model system with the data on trehalase activation.The gelatinization temperature of starch granules is shifted upwards with about 3–5 K/1000 atm (1.013×10⁵ kPa). Pressures higher than 1500 atm do not further increase the gelatinization temperature. However, no reversal of the effect, as occurs with protein conformational changes, is seen with pressure up to 2500 atm. Also for trehalase activation we find a continuous upward shift of the activation temperature with about 5–9 K/1000 atm. These data are in agreement with a thermal transition in a polysaccharide matrix, being the trigger in the heat activation of trehalase.     

Effect of pressure and temperature on the gelatinization of starch at various starch concentrations

The effects of pressure, temperature, and treatment time on the degree of gelatinization were determined with differential scanning calorimetry measurements for wheat starch-water mixtures with starch concentrations varying between 5 and 80 w/w %. Although simple models could be used to describe the degree of starch gelatinization as a function of pressure or temperature, a more complex model based on the Gibbs energy difference had to be used to describe the degree of gelatinization as a function of both pressure and temperature. The experimental and model data were used to construct a phase diagram for 5, 30, and 60 w/w % wheat starch-water mixtures. Data obtained from literature were in accordance with our phase diagrams. These phase diagrams can be used to estimate the degree of gelatinisation after applying a certain pressure and temperature on a starch-water mixture with starch concentrations in the range of 5 and 60 w/w %.     

Thermodynamic aspects of the gelatinization and swelling of crosslinked starch

Samples of epichlorohydrin crosslinked potato starch were prepared by using a high ratio of starch to water and alkali concentration below the gelatinization level. This yielded crosslinked samples that were partially crystalline, and where the number of crosslinks could be varied between 1 and 20 crosslinks per 100 anhydroglucose units. The degree of swelling varied regularly with degree of crosslinking, and the molecular weight between crosslinks M_c as derived from swelling data in a good swelling agent compared favorably with M_c derived from chemical analysis. From the heat of gelatinization of the crosslinked starches, as observed in a differential scanning calorimeter, for gelatinization in glycerol, water, and dimethylsulfoxide, a model for the gel state of the crosslinked starch is proposed. It is concluded that the entropy of melting is the determining factor in establishing the temperature of gelatinization.     

Starch Gelatinization at Different Temperatures as Measured by Enzymic Digestion Method

The gelatinization process of potato starch was isothermally investigated at 52.5∽65.3°C. The degree of gelatinization was measured by an enzymic digestion method using glucoamylase. When the starch–water suspension was incubated at a definite temperature the gelatinization reached a limit at each temperature after 30∽60 min incubation. So, it can be supposed that starch gelatinization reached an equilibrium state. It was found that gelatinization of potato starch occurred even at 52.5°C, a temperature which is lower than the so-called gelatinization temperature generally reported. Starch gelatinization was found to follow first order kinetics, and from the temperature dependence of the rate constants obtained, the activation energy was calculated to be 22±5 kcal/mol. The relationship between the degree of gelatinization of the starch whose gelatinization reached an equilibrium state at a definite temperature and the incubation temperature gave a transition curve expressed, by the fraction of gelatinized potato starch granules as a function of temperature, and the half-transition temperature was found to be 59.1°C. From the transition curve.the van’t Hoff enthalpy for gelatinization was determined to be +130±3 kcal/mol.     

Effects of structural imperfection on gelatinization characteristics of amylopectin starches with A- and B-type crystallinity

The aim of the present work was to investigate the effect of physical structures on the properties of starch granules. Starches with a high amylopectin content possessing A- and B-type crystallinity were chosen for the study. The gelatinization temperature decreased in the following order: maize (A) > potato (B) > wheat (A) > barley (A), which did not reflect a correlation with the type of crystallinity. Low values of gelatinization temperature were accompanied with high free surface energy of the crystallites. It is proposed that these data are caused by different types of imperfections in starch crystals. Annealing resulted in an enhancement of the gelatinization temperature and a decrease of the free surface energy of the crystallites for all starches reflecting a partial improvement of crystalline perfection. A limited acid hydrolysis (lintnerization) of the starches decreased the gelatinization temperature because of a partial disruption of the crystalline lamellae and an increase of the amount of defects on the edges of the crystallites. Annealing of the lintnerized starches improved the structure of maize and potato starch, giving them similar structural and physicochemical parameters, which was opposite the behavior of the annealed sample from wheat. The possible nature of removable and nonremovable defects inside the crystalline region of the starch granules is discussed. It is concluded that, besides the allomorphic A- and B-types of crystal packing, physical defects in the crystals possess a major impact on starch gelatinization.     

Starch gelatinization under pressure studied by high pressure DSC

The gelatinization process of waxy corn starch under different pressures up to 10.0 MPa was investigated using a high pressure DSC. Compressed air and carbon dioxide were used as pressure resources. Effect of pressure and annealing under pressure on gelatinization of waxy corn starch was systematically studied, in particular on the gelatinization temperature and enthalpy. The results show that the peak temperature of gelatinization was increased slightly initially then remained stable with increasing pressure. The gelatinization enthalpy was decreased under pressure processing. Annealing the starch under pressure condition, just below its gelatinization temperature, increased gelatinization temperature but kept gelatinized enthalpy constant. Morphologies of starch granules treated under pressure were studied using an optical microscope and SEM. There is no discernable difference of starch granules treated with and without pressure, which indicates the pressures are not high enough to destroy crystalline structure. The intensity of the pressure acts as a key factor to influence the gelatinization of starch rather than the nature of the gas. Effect of pressure on the multi-endotherm detected by DSC for starch with intermediate water is used to study the mechanisms. The effect of pressure can be explained by the enhancement of water diffusion in the amorphous range.     

Annealing properties of potato starches with different degrees of phosphorylation

Changes in the gelatinization temperature interval and gelatinization enthalpy with annealing time at 50 °C were followed for a number of potato starch samples, with different degrees of phosphorylation, using differential scanning calorimetry. The gelatinization temperature increased with the length of the annealing time up to the maximum time of 1280 min and a clear relation to the degree of phosphorylation was observed. The gelatinization enthalpy changed very slowly during the initial period of annealing, but faster in the later stages of the process. The increase in enthalpy was largest for the samples with the highest degree of phosphorylation. The phosphate level remained almost unaffected during the entire process. Therefore, the effects observed are not caused by hydrolysis of the phosphate esters, but rather by their reorientation toward positions causing less interference of molecular and crystalline structure of amylopectin helices.     

Kinetic modeling of sporulation and product formation in stationary phase by Bacillus coagulans RK-02 vis-à-vis other Bacilli

A logistic kinetic model was derived and validated to characterize the dynamics of a sporogenous bacterium in stationary phase with respect to sporulation and product formation. The kinetic constants as determined using this model are particularly important for describing intrinsic properties of a sporogenous bacterial culture in stationary phase. Non-linear curve fitting of the experimental data into the mathematical model showed very good correlation with the predicted values for sporulation and lipase production by Bacillus coagulans RK-02 culture in minimal media. Model fitting of literature data of sporulation and product (protease and amylase) formation in the stationary phase by some other Bacilli and comparison of the results of model fitting with those of Bacillus coagulans helped validate the significance and robustness of the developed kinetic model.     

On the effects of surface active agents on the gelatinization of starch — a calorimetric investigation

Differential scanning calorimetry (DSC) was used to study how the gelatinization process of starch is affected by the presence of surface active agents. A decrease in gelatinization enthalpy was observed, which was explained by an exothermic formation of amylose-lipid complexes during the gelatinization. It was also found that certain substances (sodium dodecyl sulphate and lysolecithin) made the gelatinization occur earlier, whereas another (sodium stearoyl lactylate) delayed the gelatinization. The results showed further that the phase behaviour of the surface active agent greatly affected the amount of complex formed, and also that the source of starch affected this amount. It was found that the complex formation occurred more easily in wheat starch than in potato starch.     

屎肠球菌产生有机酸降低琼脂胶凝作用

目的 探讨细菌破坏琼脂凝胶形成的原因和机制。方法 利用细菌培养技术培养细菌;高压灭菌再融化琼脂‒酵母培养基,室温下观察其是否凝固;利用硬度测量仪测量培养基硬度;pH测量仪测量pH值。结果 首先,固体琼脂培养基在培养屎肠球菌之后再融化出现室温条件下不凝固现象;其次,pH是一个影响琼脂凝固性的主要因素;最后,屎肠球菌通过发酵产酸而降低pH,当pH值小于4时就破坏了琼脂的凝固性。结论 细菌发酵产生酸性物质,降低了培养基pH,引起琼脂凝固点降低。     

Ionophore-induced oscillations in erythrocytes. A kinetic model

The kinetic model for K+, H+, Ca2+ concentrational self-oscillations in erythrocytes induced by A23187 and FCCP ionophores are considered. The model is based on the experimental data available and contains the minimal number of essential variables. The model was analysed by the method based on the graph representation of kinetic equations. The critical graph fragments provoking self-oscillatory trajectories in the system were revealed. It is shown that self-oscillatory behaviour is basically induced by conjugated processes produced by A23187. The parameter domain for self-oscillations is estimated including parameters of Ca2+-induced (through K+-channels) proton transport with FCCP participation. Numerical integration of kinetic equations was performed. The results obtained are in a good qualitative agreement with experimental data.     

Modeling, simulation, and kinetic analysis of a heterogeneous reaction system for the enzymatic conversion of poorly soluble substrate.

We developed a kinetic model that describes a heterogeneous reaction system consisting of a solid substrate suspension for the production of D-amino acid using D-hydantoinase. As a biocatalyst, mass-produced free and whole cell enzymes were used. The heterogeneous reaction system involves dissolution of a solid substrate, enzymatic conversion of the dissolved D-form substrate, spontaneous racemization of an L-form substrate to D-form, and deactivation of the enzyme. In the case of using whole cell enzymes, transfer of the dissolved substrate and product through the cell membrane was considered. The kinetic parameters were determined from experiments, literature data, and by using Marquardt's method of nonlinear regression analysis. The model was simulated using the kinetic parameters and compared with experimental data, and a good agreement was observed between the experimental results and the simulation ones. Factors affecting the kinetics of the heterogeneous reaction system were analyzed on the basis of the kinetic model, and the efficiency of the reaction systems using free and whole cell enzymes was also compared.     

A concavity property of the viscosity growth curve during alkali gelatinization of rice starch

A qualitative difference between the viscosity–time curves for thermal and strong alkali gelatinization of rice starch was demonstrated using continuous capillary viscometry. During the thermal (60, 70, 75, 80 °C) gelatinization with distilled water, the viscosity growth curves kept a convexity property, in accordance with the past known results. In contrast, the viscosity growth curves for the cold (15, 20 °C) gelatinization with a 0.146 N NaOH solution showed a concavity property in the first half of whole gelatinization process. This result confirmed our previous result having been obtained from batch-type measurement with use of a cone-plate viscometer. On the basis of the first-order reaction hypothesis for gelatinization degree, this novel viscosity growth behavior in cold alkali gelatinization could be described in terms of the mixing rule of viscosity distinct from that had been applied to thermal gelatinization.     

k-Cone analysis: determining all candidate values for kinetic parameters on a network scale

The absence of comprehensive measured kinetic values and the observed inconsistency in the available in vitro kinetic data has hindered the formulation of network-scale kinetic models of biochemical reaction networks. To meet this challenge we present an approach to construct a convex space, termed the k-cone, which contains all the allowable numerical values of the kinetic constants in large-scale biochemical networks. The definition of the k-cone relies on the incorporation of in vivo concentration data and a simplified approach to represent enzyme kinetics within an established constraint-based modeling approach. The k-cone approach was implemented to define the allowable combination of numerical values for a full kinetic model of human red blood cell metabolism and to study its correlated kinetic parameters. The k-cone approach can be used to determine consistency between in vitro measured kinetic values and in vivo concentration and flux measurements when used in a network-scale kinetic model. k-Cone analysis was successful in determining whether in vitro measured kinetic values used in the reconstruction of a kinetic-based model of Saccharomyces cerevisiae central metabolism could reproduce in vivo measurements. Further, the k-cone can be used to determine which numerical values of in vitro measured parameters are required to be changed in a kinetic model if in vivo measured values are not reproduced. k-Cone analysis could identify what minimum number of in vitro determined kinetic parameters needed to be adjusted in the S. cerevisiae model to be consistent with the in vivo data. Applying the k-cone analysis a priori to kinetic model development may reduce the time and effort involved in model building and parameter adjustment. With the recent developments in high-throughput profiling of metabolite concentrations at a whole-cell scale and advances in metabolomics technologies, the k-cone approach presented here may hold the promise for kinetic characterization of metabolic networks as well as other biological functions at a whole-cell level.     

Acid hydrolysis of native and annealed starches and branch-structure of their Naegeli dextrins

Eight commercial starches, including common corn, waxy corn, wheat, tapioca, potato, Hylon V, Hylon VII, and mung bean starch, were annealed by a multiple-step process, and their gelatinization characteristics were determined. Annealed starches had higher gelatinization temperatures, reduced gelatinization ranges, and increased gelatinization enthalpies than their native starches. The annealed starches with the highest gelatinization enthalpies were subjected to acid hydrolysis with 15.3% H2SO4, and Naegeli dextrins were prepared after 10 days' hydrolysis. Annealing increased the acid susceptibility of native starches in the first (rapid) and the second (slow) phases with potato starch showing the greatest and high amylose starches showing the least changes. Starches with a larger shift in onset gelatinization temperature also displayed a greater percent hydrolysis. The increase in susceptibility to acid hydrolysis was proposed to result from defective and porous structures that resulted after annealing. Although annealing perfected the crystalline structure, it also produced void space, which led to porous structures and possible starch granule defects. The molecular size distribution and chain length distribution of Naegeli dextrins of annealed and native starches were analyzed. The reorganization of the starch molecule during annealing occurred mainly within the crystalline lamellae. Imperfect double helices in the crystalline lamellae improved after annealing, and the branch linkages at the imperfect double helices became protected by the improved crystalline structure. Therefore, more long chains were observed in the Naegeli dextrins of annealed starches than in native starches.     

Kinetic behaviour of free lipase and mica-based immobilized lipase catalyzing the synthesis of sugar esters

The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.     

Kinetic study of the enzymatic cycling reaction conducted with 3alpha-hydroxysteroid dehydrogenase in the presence of excessive thio-NAD(+) and NADH

We have established a simple kinetic model applicable to the enzyme cycling reaction for the determination of 3alpha-hydroxysteroids. This reaction was conducted under the reversible catalytic function of a single 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) with nucleotide cofactors, thio-NAD(+) (one of the NAD(+) analogues) for the oxidation of 3alpha-hydroxysteroids and NADH for the reduction of 3-oxosteroids. This model was constructed based on the reaction mechanism of 3alpha-HSD, following an ordered bi-bi mechanism with cofactor binding first, under the assumption that the respective enzyme-cofactor complexes were distributed according to the initial ratio of thio-NAD(+) to NADH by the rapid equilibrium of both enzyme-cofactor complexes. The cycling rate in the new kinetic model could be expressed with the dissociation constants of enzyme-cofactor complexes and the initial concentrations of cofactors and enzyme. The cycling rate was verified by a comparison with the experimental data using 3alpha-HSD from Pseudomonas sp. B-0831. The results showed that the experimental data corresponded well with the results obtained from the kinetic model.     

Development of a phenomenological modeling approach for prediction of growth and xanthan gum production using Xanthomonas campestris

An unstructured kinetic model for xanthan production is described and fitted to experimental data obtained in a stirred batch reactor. The culture medium was composed of several nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) consumed sequentially. The model proposed is able to describe this sequential consumption of nitrogen sources, the consumption of inorganic phosphate and carbon, the evolution of biomass, and production of xanthan. The parameter estimation has been performed by fitting the kinetic model in differential form to experimental data. Runs of the model for simulating xanthan gum production as a function of the initial concentration of inorganic phosphate have shown the positive effect of phosphate limitation on xanthan yield, though diminishing rates of production. The model was used to predict the kinetic parameters for a medium containing a 2-fold lower initial phosphate concentration. When tested experimentally, the measured fermentation parameters were in close agreement with the predicted model values, demonstrating the validity of the model.     

State diagram of potato starch–water mixtures treated with high hydrostatic pressure

Potato starch–water mixture was treated with high hydrostatic pressure (HHP) of up to 1.2 GPa, and effect of starch content (10–70% (w/w)) on HHP-gelatinization was investigated by differential scanning calorimetry (DSC). Depending on the treatment pressure and potato starch content, DSC thermograms showed decrease in enthalpy change of heat gelatinization reflecting the progress of HHP-gelatinization and increase in enthalpy change of re-gelatinization of retrograded starch. From the viewpoint of the enthalpy changes, physically modified state of HHP-treated potato starch–water mixtures was classified as follows: no change, partial gelatinization, complete gelatinization, partial gelatinization and retrogradation, and complete gelatinization and retrogradation. A state diagram of potato starch–water mixtures (treatment pressure vs. starch content) was presented.