This diploma thesis is focused on the determination of distribution a surfactant at phase separation in polymer-surfactant system. Sodium hyaluronate of three molecular weights was chosen as a polymer, CTAB was used as a surfactant. The experiments were performed in an environment of 0,15 M NaCl. The measurement of the CTAB concentration in samples was based on the formation of coloured complexes of CTAB and picric acid in chloroform. UV-VIS spectroscopy was used for the detection of these complexes. The content of CTAB was determined in four gels of different composition (2% Hya + 200 mM CTAB, 2 % Hya + 50 mM CTAB, 0,5 % Hya + 200 mM CTAB, 0,5 % Hya + 50 mM CTAB). All gels were gradually washed five times with 0,15 M NaCl. The content of CTAB was determined for newly prepared gels and for gels after 2 and 4 months since the preparation. Initial concentrations of CTAB and hyaluronan have greater influence on the distribution of CTAB after phase separation, than the molecular weight of hyaluronan. The content of CTAB in newly prepared gels and after 4 months since preparation is very similar and demonstrates the stability of the system.

Konjac glucomannan (KGM), the main biologically active constituent of konjac flour extracted from corms of Amorphophallus konjac (konjac), can be used to prepare functional foods and may also have potential as a pharmaceutical product to combat obesity. The current study employed three experimental approaches to study the biology and chemistry of konjac, namely (1) glasshouse experiments to study the morphogenesis, growth and productivity of konjac plants, (2) a histological and immunocytochemical investigation of the localisation and developmental regulation of the deposition and metabolism of KGM in developing corm tissues, and (3) a comparative study of methodologies for the extraction and analysis of KGM. The current data demonstrated a morphological and functional separation between the ventral and dorsal regions of corms. The ventral region appeared to function as a source during the initial period of shoot development, while the dorsal region appeared to operate as a sink after the development of mature canopy. Once the corm reached maturity, both an inflorescence and a leaf were produced within a single season. It has also been demonstrated that the age of the ‘mother’ corm is an important factor affecting the quality of offsets produced. An anti-mannan antiserum detected a temporally regulated pattern of mannan epitope production within glucomannan idioblasts in developing corm tissues, with increased expression as the corm approached maturity/dormancy. The current observations also suggest that the mobilization of KGM initiates at the periphery of the corm and proceeds inwards towards the centre of the corm. Compositional analysis showed that the purified konjac flour (PKF) produced using a modified extraction procedure contained 92% glucomannan, with a weight average molecular weight (Mw), polydispersity index (PDI) and degree of acetylation (DA) of 9.5 ± 0.6 x 105 gmol-1, 1.2 and 2.8 wt. %. These data, plus Fourier-transform infrared spectral (FTIR) and zero shear viscosity analyses of the extract (PKF) were all consistent with the literature. Comparison of three existing methodologies for the quantitative analysis of the KGM content, namely 3,5-dinitrosalicylic acid (3,5-DNS), phenol-sulphuric acid and enzymatic colorimetric assays; indicated that the 3,5-DNS colorimetric assay was the most reproducible and accurate method, with a linear correlation coefficient of 0.997 and recoveries between 97% and 103% across three spiking levels of starch. In summary, this study has provided a better understanding of aspects of the biology and cultivation of A. konjac and has also produced methodologies which can be used as the basis for an improved good laboratory practice (GLP) for the commercial extraction and analysis of this multifunctional natural polymer.

Kinetic inactivation of factor XIIIa and MTG were performed in a pressure range from 0.1 to 400 MPa at 40°C within a time from 0 to 60 min in a TRIS-acetate buffer at pH 6.0. The inactivation of both enzymes at these conditions followed a first order reaction model. The high inactivation rate constant of 26.6 x10-3/min-1 for factor XIIIa at low pressure (50 MP) indicated that this enzyme is much easier to inactivate than MTG, which achieved an inactivation rate constant value of 9.7 x10-3/min at higher pressure (200 MPa). An inactivation volume of –10.17±0.5 cm3/mol confirmed that MTG is very stable under high pressure. The stability of MTG under high pressure and thermal treatment was related to its conformational changes. Enzyme inactivation was accompanied by secondary and tertiary structure changes until an irreversible protein precipitation is achieved. The tertiary structure, represented by circular dichroism spectra in the aromatic region showed differences among native and MTG samples treated under high pressure, as well as at elevated temperature. Tyrosine bands, indicating protein unfolding, increased proportionally with increasing pressure treatment above 400 MPa. Nevertheless, compared to pressure, a maximal enhancement could be observed after thermal treatment at 0.1 MPa at 80°C. That demonstrated the exposure of hydrophobic groups to the protein surface with a concomitant protein unfolding. The spectra in the far ultraviolet region showed that increasing high pressure and high temperature lead to alterations in the secondary structure. The mathematical algorithms CONTIN used to calculate secondary structures stated that the 24.5% of alpha-helix of native MTG decreased to 17.2% after a treatment at 400 MPa at 40°C for 60 min and to 6.5% after a treatment at 0.1 MPa at 80°C for 2 min. However, beta-strand structures remained relatively stable after these several treatments. MTG is arranged in a way that the active site is located between beta-strand domains that are surrounded by alpha-helices, the results of this investigation suggested that MTG activity is related with the relative stability of alpha-helix and the outstanding stability of the central beta-strand structure. The irreversible precipitated protein observed at 600 MPa at 40°C for 60 min and 0.1 MPa at 80°C for 2 min was caused principally by the formation of disulfides bonds, because high pressure and high thermal treatment lead to the exposition of the Cys64 residue towards the solvent with the subsequent ability to react with neighbouring cysteine residues. Furthermore, the reaction between protein and reducing sugars resulted in the formation of Maillard products. Furosine, as an indicator of the early stages of Maillard reaction was measured. Concentration values of 261.0 mg/g protein from samples treated at 600 MPa and 40°C and 238.5 mg/g protein from samples treated at and 0.1 MPa and 80°C for 2 min were obtained. Pentosidine a subsequent product observed in the advanced Maillard reaction was also present. Concentrations of 13.7 and 6.7 mg/g protein were obtained in the samples treated at 600 MPa and 40°C for 60 min and 0.1 MPa and 80°C for 2 min, respectively. Kinetic inactivation studies of MTG in a pressure range from 0.1 to 600 MPa at 10, 30, 40, and 50°C within a long time range from 0 to 140 h were performed in order to study MTG stability under the simultaneous effect of pressure and temperature. The inactivation kinetic showed a first and very fast step and a second very slow step suggesting irreversible inactivation behaviour. Activation energy and entropy difference decreased with increasing pressure. Thereby, the inactivation rate constants of enzyme were less temperature dependent at high pressure. The effect of pressure and temperature on MTG inactivation had a synergistic behaviour. At temperatures of 10, 30, and 40°C, increasing pressure leads to increasing inactivation rate constants. However at 50°C a tendency change occurred. Negative activation volumes of –16.2±0.5, -13.6±0.1, -11.2±0.3 cm3/mol were obtained for 10, 30 and 40°C respectively and for treatment at 50°C a positive value of about +3.0±2.0 cm3/mol in a pressure range from 0.1 to 300 and a negative volume of –11.0±0.4 cm3/mol MPa from 300 to 600 MPa were calculated. A pressure/temperature diagram from inactivation rate constants was performed to represent MTG stability. The diagram shows that in a pressure and temperature range from 0.1 to 550 MPa and 10 to 40°C, pressure induces MTG stabilization against heat denaturation. At 50°C in range from 0.1 to 300 MPa, pressure induces also enzyme stabilization again heat denaturation, but at the same temperature and above 300 MPa the enzyme was inactivated. After MTG stability analysis, reaction kinetics from MTG with individual caseins in a TRIS-acetate buffer pH 6.0 were performed under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40°C. The reaction was monitored by gel permeation chromatography under in three assumptions: 1) The initial velocity kinetics was obtained from a non-progressive enzymatic reactions with the products. 2) The substrate concentration exceeded enzyme concentration. 3) The sum of the individual catalytic constants of the reactive glutamine residues inside caseins are represented by a single MTG-monomeric casein complex. Enzyme reaction kinetics of MTG with the individual caseins carried out at 0.1 MPa at 40°C showed Michaelis-Menten-Henri behaviour with maximal velocities of 2.7 x 10-3, 0.8 x 10-3, and 1.3 x 10-3 mmol/L∙min and Km values of 59 x 10-3, 64 x 10-3 and 50 x 10-3 mmol/L of beta-, alpha-s1-, and whole-casein, respectively. This suggested that MTG achieved a maximal velocity with ß-casein, but had the best affinity with acid casein followed by beta- casein and finally alpha-s1-casein. Enzyme reaction kinetics of beta-casein carried out at 400 MPa and 40°C also showed a Michaelis-Menten-Henri behaviour with a similar maximal velocity of 2.6 x 10-3 mmol/L×min, but the Km value of 144 x 10-3 mmol/L showing kinetical similarity to a non-competitive inhibition. The reaction of MTG with alpha-s1-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics. Kinetic parameters showed that the affinity of MTG to beta- and alpha-s1-casein under atmospheric pressure is higher than the affinity of MTG to these caseins under high pressure. This loss of affinity can be explained by a constant number of reactive glutamine residues of casein, although the protein is unfolding at high pressure, a decrease of enzyme activity of MTG to 74% after treatment at 400 MPa at 40°C for 15 min and self association of casein under thermal and high pressure treatment. Fur technological application, the formation of acid milk gels was studied under the influence of MTG within its range of pH stability. Simultaneous addition of MTG and different concentrations of glucono-delta-lactone (Gdl) to casein solutions (5% w/v) at 40°C was analysed. Gels firmness was accessed by oscillation rheometry and gel permeation chromatography. Oscillation rheometry data showed that the time of gelation decreased with an increasing Gdl concentration added to the system, however higher concentrations of Gdl caused the formation of weaker gels. Addition of 1 g Gdl/g protein without MTG caused gelation within 5 min and a storage module value G´ of 48.9 Pa. With the simultaneous addition of 1 g Gdl/g protein and 6 U MTG/ g protein the gelation time was 4 min and the reached storage modulus was 63.7 Pa. However, the addition of 0.21 g Gdl/g protein and 6 U/g protein MTG increase the gelation time to about 69 min, but, a higher module value G´ of 111.0 Pa was achieved. Addition of high Gdl concentration caused a rapid drop of pH below 5 leading to a fast enzyme inactivation. However addition of very low Gdl concentrations was also not optimal. The simultaneous influence of MTG and Gdl concentration on the gelation time and elastic properties was evaluated by a central composite rotatable design (CCRD). The resulting quadratic storage modulus model showed that, MTG concentration had a significant influence on storage modulus G´ and, that the firmness of the gels increase in direct proportion with MTG activity with the existence of a optimum Gdl concentration, whereas the resulting linear model of the gelation time stated that Gdl concentration has a significant influence on the gelation time, while it is independent of the MTG activity. A maximal firmness of 136 ± 2 Pa was reached between a range of 0.24 - 0.27 g Gdl/g protein and 5.8 U MTG/g within a time from 49 to 59 min. Gel permeation chromatography analysis demonstrated that acid gels induced by Gdl were formed by reversible cross-linking like electrostatic interactions and hydrogen bonds as well as disulfide bonds caused by temperature treatment. Whereas, the addition of MTG proved the formation of non-reversible cross-linking like oligomers based on Ne-(g-glutamyl)- lysine, which gave more firmness and stabilization on the casein gel network.

Kinetic inactivation of factor XIIIa and MTG were performed in a pressure range from 0.1 to 400 MPa at 40°C within a time from 0 to 60 min in a TRIS-acetate buffer at pH 6.0. The inactivation of both enzymes at these conditions followed a first order reaction model. The high inactivation rate constant of 26.6 x10-3/min-1 for factor XIIIa at low pressure (50 MP) indicated that this enzyme is much easier to inactivate than MTG, which achieved an inactivation rate constant value of 9.7 x10-3/min at higher pressure (200 MPa). An inactivation volume of –10.17±0.5 cm3/mol confirmed that MTG is very stable under high pressure. The stability of MTG under high pressure and thermal treatment was related to its conformational changes. Enzyme inactivation was accompanied by secondary and tertiary structure changes until an irreversible protein precipitation is achieved. The tertiary structure, represented by circular dichroism spectra in the aromatic region showed differences among native and MTG samples treated under high pressure, as well as at elevated temperature. Tyrosine bands, indicating protein unfolding, increased proportionally with increasing pressure treatment above 400 MPa. Nevertheless, compared to pressure, a maximal enhancement could be observed after thermal treatment at 0.1 MPa at 80°C. That demonstrated the exposure of hydrophobic groups to the protein surface with a concomitant protein unfolding. The spectra in the far ultraviolet region showed that increasing high pressure and high temperature lead to alterations in the secondary structure. The mathematical algorithms CONTIN used to calculate secondary structures stated that the 24.5% of alpha-helix of native MTG decreased to 17.2% after a treatment at 400 MPa at 40°C for 60 min and to 6.5% after a treatment at 0.1 MPa at 80°C for 2 min. However, beta-strand structures remained relatively stable after these several treatments. MTG is arranged in a way that the active site is located between beta-strand domains that are surrounded by alpha-helices, the results of this investigation suggested that MTG activity is related with the relative stability of alpha-helix and the outstanding stability of the central beta-strand structure. The irreversible precipitated protein observed at 600 MPa at 40°C for 60 min and 0.1 MPa at 80°C for 2 min was caused principally by the formation of disulfides bonds, because high pressure and high thermal treatment lead to the exposition of the Cys64 residue towards the solvent with the subsequent ability to react with neighbouring cysteine residues. Furthermore, the reaction between protein and reducing sugars resulted in the formation of Maillard products. Furosine, as an indicator of the early stages of Maillard reaction was measured. Concentration values of 261.0 mg/g protein from samples treated at 600 MPa and 40°C and 238.5 mg/g protein from samples treated at and 0.1 MPa and 80°C for 2 min were obtained. Pentosidine a subsequent product observed in the advanced Maillard reaction was also present. Concentrations of 13.7 and 6.7 mg/g protein were obtained in the samples treated at 600 MPa and 40°C for 60 min and 0.1 MPa and 80°C for 2 min, respectively. Kinetic inactivation studies of MTG in a pressure range from 0.1 to 600 MPa at 10, 30, 40, and 50°C within a long time range from 0 to 140 h were performed in order to study MTG stability under the simultaneous effect of pressure and temperature. The inactivation kinetic showed a first and very fast step and a second very slow step suggesting irreversible inactivation behaviour. Activation energy and entropy difference decreased with increasing pressure. Thereby, the inactivation rate constants of enzyme were less temperature dependent at high pressure. The effect of pressure and temperature on MTG inactivation had a synergistic behaviour. At temperatures of 10, 30, and 40°C, increasing pressure leads to increasing inactivation rate constants. However at 50°C a tendency change occurred. Negative activation volumes of –16.2±0.5, -13.6±0.1, -11.2±0.3 cm3/mol were obtained for 10, 30 and 40°C respectively and for treatment at 50°C a positive value of about +3.0±2.0 cm3/mol in a pressure range from 0.1 to 300 and a negative volume of –11.0±0.4 cm3/mol MPa from 300 to 600 MPa were calculated. A pressure/temperature diagram from inactivation rate constants was performed to represent MTG stability. The diagram shows that in a pressure and temperature range from 0.1 to 550 MPa and 10 to 40°C, pressure induces MTG stabilization against heat denaturation. At 50°C in range from 0.1 to 300 MPa, pressure induces also enzyme stabilization again heat denaturation, but at the same temperature and above 300 MPa the enzyme was inactivated. After MTG stability analysis, reaction kinetics from MTG with individual caseins in a TRIS-acetate buffer pH 6.0 were performed under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40°C. The reaction was monitored by gel permeation chromatography under in three assumptions: 1) The initial velocity kinetics was obtained from a non-progressive enzymatic reactions with the products. 2) The substrate concentration exceeded enzyme concentration. 3) The sum of the individual catalytic constants of the reactive glutamine residues inside caseins are represented by a single MTG-monomeric casein complex. Enzyme reaction kinetics of MTG with the individual caseins carried out at 0.1 MPa at 40°C showed Michaelis-Menten-Henri behaviour with maximal velocities of 2.7 x 10-3, 0.8 x 10-3, and 1.3 x 10-3 mmol/L∙min and Km values of 59 x 10-3, 64 x 10-3 and 50 x 10-3 mmol/L of beta-, alpha-s1-, and whole-casein, respectively. This suggested that MTG achieved a maximal velocity with ß-casein, but had the best affinity with acid casein followed by beta- casein and finally alpha-s1-casein. Enzyme reaction kinetics of beta-casein carried out at 400 MPa and 40°C also showed a Michaelis-Menten-Henri behaviour with a similar maximal velocity of 2.6 x 10-3 mmol/L×min, but the Km value of 144 x 10-3 mmol/L showing kinetical similarity to a non-competitive inhibition. The reaction of MTG with alpha-s1-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics. Kinetic parameters showed that the affinity of MTG to beta- and alpha-s1-casein under atmospheric pressure is higher than the affinity of MTG to these caseins under high pressure. This loss of affinity can be explained by a constant number of reactive glutamine residues of casein, although the protein is unfolding at high pressure, a decrease of enzyme activity of MTG to 74% after treatment at 400 MPa at 40°C for 15 min and self association of casein under thermal and high pressure treatment. Fur technological application, the formation of acid milk gels was studied under the influence of MTG within its range of pH stability. Simultaneous addition of MTG and different concentrations of glucono-delta-lactone (Gdl) to casein solutions (5% w/v) at 40°C was analysed. Gels firmness was accessed by oscillation rheometry and gel permeation chromatography. Oscillation rheometry data showed that the time of gelation decreased with an increasing Gdl concentration added to the system, however higher concentrations of Gdl caused the formation of weaker gels. Addition of 1 g Gdl/g protein without MTG caused gelation within 5 min and a storage module value G´ of 48.9 Pa. With the simultaneous addition of 1 g Gdl/g protein and 6 U MTG/ g protein the gelation time was 4 min and the reached storage modulus was 63.7 Pa. However, the addition of 0.21 g Gdl/g protein and 6 U/g protein MTG increase the gelation time to about 69 min, but, a higher module value G´ of 111.0 Pa was achieved. Addition of high Gdl concentration caused a rapid drop of pH below 5 leading to a fast enzyme inactivation. However addition of very low Gdl concentrations was also not optimal. The simultaneous influence of MTG and Gdl concentration on the gelation time and elastic properties was evaluated by a central composite rotatable design (CCRD). The resulting quadratic storage modulus model showed that, MTG concentration had a significant influence on storage modulus G´ and, that the firmness of the gels increase in direct proportion with MTG activity with the existence of a optimum Gdl concentration, whereas the resulting linear model of the gelation time stated that Gdl concentration has a significant influence on the gelation time, while it is independent of the MTG activity. A maximal firmness of 136 ± 2 Pa was reached between a range of 0.24 - 0.27 g Gdl/g protein and 5.8 U MTG/g within a time from 49 to 59 min. Gel permeation chromatography analysis demonstrated that acid gels induced by Gdl were formed by reversible cross-linking like electrostatic interactions and hydrogen bonds as well as disulfide bonds caused by temperature treatment. Whereas, the addition of MTG proved the formation of non-reversible cross-linking like oligomers based on Ne-(g-glutamyl)- lysine, which gave more firmness and stabilization on the casein gel network.