Assessment of the usefulness of bacterial cellulose produced by Gluconacetobacter xylinus E25 as a new biological implant.

Bionanocellulose (BNC) is a clear polymer produced by the bacterium Gluconacetobacter xylinus. In our current study, "Research on the use of bacterial nanocellulose (BNC) in regenerative medicine as a function of the biological implants in cardiac and vascular surgery", we carried out material analysis, biochemical analysis, in vitro tests and in vivo animal model testing. In stage 1 of the project, we carried out physical and biological tests of BNC. This allowed us to modify subsequent samples of bacterial bionanocellulose. Finally, we obtained a sample that was accepted for testing on an animal model. That sample we define BNC1. Patches of BNC1 were then implanted into pigs' vessel walls. During the surgical procedures, we evaluated the technical aspects of sewing in the bioimplant, paying special attention to bleeding control and tightness of the suture line and the BNC1 bioimplant itself. We carried out studies evaluating the reaction of an animal body to an implantation of BNC1 into the circulatory system, including the general and local inflammatory reaction to the bioimplant. These studies allowed us to document the potential usefulness of BNC as a biological implant of the circulatory system and allowed for additional modifications of the BNC to improve the properties of this new implantable biological material.

The Effect of High Pressure and Subzero Temperature on Gelation of Washed Cod and Salmon Meat.

The objective of the present work is to examine the influence of pressure up to 193 MPa at subzero temperature (without freezing of water) on myofibrillar proteins of salmon and cod meat and on the properties of gels obtained from washed mince of these fish. The solubility of proteins from myofibrils of cod and salmon meat suspended in 100 mM KCl solution increased after treating the samples with pressure above 60 MPa. The results of SDS- -PAGE analysis showed that under these conditions two myosin light chains, tropomyosin and troponin T were released from myofibrils. The solubility of proteins in 0.9 M NaCl solution of washed fish meat after pressure treatment at 60 MPa and -5 °C decreased to about 80-90% and at 193 MPa and -20 °C to 60%. Pressurization of cod meat decreased only slightly the solubility of proteins in SDS and urea solution and the solubility of salmon meat was similar to that in the unpressurized sample. There were no differences in the electrophoretic pattern of proteins from untreated and pressurized cod and salmon meat in the range of 60 to 193 MPa and -5 to -20 °C. The pressure treatment of washed salmon and cod meat at a temperature below 0 °C induced gelation; on the other hand, hardness of gels was lower by 28 and 26%, respectively, than that of gels formed by heating. The salmon and cod gels pressurized at 193 MPa and -20 °C and then heated were much harder than only pressurized or heated gels.

The effect of high pressure at subzero temperature on proteins solubility, drip loss and texture of fish (cod and salmon) and mammal's (pork and beef) meat.

One of the possibilities of using high-pressure technique in inactivation of microorganism is conducting this process at subzero temperature. However, for its practical application in meat preservation the appropriate properties of meat should be maintained. Therefore, the aim of this work was to examine the effect of pressure at subzero temperature (without freezing of water) on proteins and texture of mammal's and cold-adapted fish meat. The data showed that cod and salmon meat proteins were more susceptible to pressure-induced denaturation/aggregation than beef and pork proteins. Glucose and saccharose exerted protective effect on fish meat proteins treated with pressure of 111 MPa(tc) and -10 degrees C but not at 193 MPa(tc) and -20 degrees C. The pressure treatment under the latter conditions increased cook loss of fish meat but not of mammal's meat. However, after cooking the hardness of all kinds of pressurized meat was at the same level as that for unpressurized cooked samples.

Interactions of fish gelatin and chitosan in uncrosslinked and crosslinked with EDC films: FT-IR study.

Films based on fish gelatin, chitosan and blend of fish gelatin and chitosan before and after cross-linking with EDC have been characterized by FT-IR spectroscopy. The FT-IR spectrum of fish gelatin film showed the characteristic amide I, amide II and amide III bands, and the FT-IR spectrum of chitosan film confirmed that the polymer was only a partially deacetylated product, and included CH3-C=O and NH2 groups, the latter both in their free -NH2 and protonated -NH3(+) form. Analysis of FT-IR spectra of two-component, fish gelatin-chitosan film revealed the formation not only of hydrogen bonds within and between chains of polymers, but also of electrostatic interactions between -COO(-) of gelatin and -NH3(+) of chitosan. Modification with EDC provided cross-linking of composites of the film. New iso-peptide bonds formed between activated carboxylic acid groups of glutamic or aspartic acid residue of gelatin and amine groups of gelatin or/and chitosan.

Changes in bacterial cells induced by high pressure at subzero temperature.

The aim of this study was to examine the effect of pressure treatment at 193MPa and -20°C on membrane damage, changes in activity of membrane-bound ATPases and degradation of nucleic acids. The experiments were carried out with three Escherichia coli strains, in the exponential and stationary phases of growth, and differing in sensitivity to pressure. All E. coli strains subjected to pressure in the exponential phase of growth were inactivated by 6 log cycles, independently of the strain, which was accompanied by a total loss of ability to plasmolyse, an increase in irreversible membrane permeability to PI, and a reduction of cellular ATP by more than 80%. After pressure treatment of stationary phase cells, the relationship between the inactivation level and the ability to plasmolyse was not as evident as in the case of exponential phase cells. Pressure treatment of two strains of E. coli K-12 and Ec160/59 in the stationary phase that decreased viability by no more than one log cycle led only to reversible permeabilization of bacterial membranes, while irreversible permeabilization was observed in the pressure sensitive E. coli IBA72 strain phase that was inactivated by 4.6 log cycles. The reduction of ATP and changes in ATPase activity after pressure treatment of tested E. coli strains in the stationary phase of growth depended on the stage of inactivation of the particular strain. Electrophoretic analysis showed degradation of RNA isolated after pressure treatment from cells of all E. coli strains tested in the exponential phase of growth. The changes of RNA induced by pressure were not visible in the case of cells in the stationary phase. The degradation of DNA isolated from pressure treated E. coli strains from the exponential as well as from the stationary phase of growth was not observed.

Effect of lysozyme or nisin on survival of some bacteria treated with high pressure at subzero temperature.

The aim of this work was to examine the inactivation of some Gram-positive and Gram-negative bacteria exposed to the pressure of 193 MPa at -20 °C in the presence of lysozyme or nisin at concentration of 400 µg/ml. The highest effect of pressure at subzero temperature and lysozyme was found with pressure sensitive Pseudomonas fluorescens; viable cells of this strain were not detected in 1 ml of sample after combined treatment. The action of pressure at subzero temperature and lysozyme or nisin against Escherichia coli led to synergistic reduction by 0.7 or 1.6 log cycles, respectively, while it was practically insignificant for two Staphylococcus aureus strains. Viability loss of E. coli and S. aureus occurred during storage for 20 h of the samples at 37 and 5 °C, which were previously pressurized with lysozyme or nisin. The synergistic effect of pressure and nisin at pH 5 against E. coli cells just after the pressure treatment was lower than that at pH 7, however, the extent of the lethal effect after storage was higher.