Degree Of Cell Disruption Research Articles

Evaluating microalgal cell disruption upon ultra high pressure homogenization

The impact of (ultra) high pressure homogenization on the degree of cell disruption was investigated for Nannochloropsis sp. suspensions. The degree of cell disruption was studied by combining four evaluation methods: turbidity measurement, scanning electron microscopy, hexane:isopropanol extraction efficiency, and fluorescence microscopy using the viability stain SYTOX green. Applying an ultra high pressure of 250 MPa obviously reduced the number of homogenization passes required to obtain a specific degree of cell disruption compared to 100 MPa. However, heating of the sample occurred at 250 MPa, resulting in extensive aggregate formation of the released intracellular material after multiple homogenization passes. Furthermore, cell wall integrity was not necessarily linked to membrane integrity, implying that moderate (U)HPH conditions are possibly sufficient for certain applications by damaging the cell membrane, without achieving full rupture of the cell wall. Once again it was proven that different methods for evaluation of cell disruption should be combined to get comprehensive insight into the disruption of microalgae.

Microalgal biomass as a (multi)functional ingredient in food products: Rheological properties of microalgal suspensions as affected by mechanical and thermal processing

Microalgae show great potential for use as novel ingredients in food products, as they are rich in several nutritional and health-beneficial components. However, addition of total microalgal biomass might alter the structural properties of the food system. Therefore, information is required about their rheological characteristics towards selection of microalgae species for specific food products. This study comprises the rheological characterization of seven commercially available microalgae species in aqueous suspensions, before and after mechanical and thermal processing. Substantial differences in rheological properties were observed between the investigated microalgal suspensions. Among the untreated suspensions, Porphyridium cruentum, Chlorella vulgaris and Odontella aurita showed the largest structural properties and could be described as weak gels. All suspensions showed shear-thinning flow behavior at the examined concentration of 8% w/w, except for Nannochloropsis species. Shear-thinning behavior was also observed for the separated serum phase of P. cruentum, which might be attributed to the presence of sulfated exopolysaccharides. During processing, rheological properties were significantly altered. High pressure homogenization was used as a mechanical treatment, followed by a pasteurization or sterilization process. Whereas suspensions of Arthrospira platensis and C. vulgaris showed an increased storage modulus and viscosity after processing, the opposite was observed for P. cruentum and O. aurita. No clear effect of processing was observed for suspensions of Nannochloropsis sp., Schizochytrium sp. and Phaeodactylum tricornutum. Investigation of the microstructure revealed differences in degree of cell disruption by high pressure homogenization, with Nannochloropsis sp. being the most resistant. Subsequent thermal processing resulted in aggregation of released cell material and/or intact cells. In conclusion, the obtained results provide the scientific knowledge base for the selection of microalgae species towards food applications. Whereas some microalgae species hardly affect the structural properties of the food product, other microalgae species show large potential for use as a structuring agent in food applications.

Ultrasound assisted methods for enhanced extraction of phycobiliproteins from marine macro-algae, Gelidium pusillum (Rhodophyta)

Extraction of phycobiliproteins (R-phycoerythrin, R-PE and R-phycocyanin, R-PC) from macro-algae is difficult due to the presence of large polysaccharides (agar, cellulose etc.) present in the cell wall which offer major hindrance for cell disruption. The present study is aimed at developing most suitable methodology for the primary extraction of R-PE and R-PC from marine macro-algae, Gelidium pusillum(Stackhouse) Le Jolis. Such extraction of phycobiliproteins by using ultrasonication and other conventional methods such as maceration, maceration in presence of liquid nitrogen, homogenization, and freezing and thawing (alone and in combinations) is reported for the first time. Standardization of ultrasonication for different parameters such as ultrasonication amplitude (60, 90 and 120µm) and ultrasonication time (1, 2, 4, 6, 8 and 10mins) at different temperatures (30, 35 and 40°C) was carried out. Kinetic parameters were estimated for extraction of phycobiliproteins by ultrasonication based on second order mass transfer kinetics. Based on calorimetric measurements, power, ultrasound intensity and acoustic power density were estimated to be 41.97W, 14.81W/cm2 and 0.419W/cm3, respectively. Synergistic effect of ultrasonication was observed when employed in combination with other conventional primary extraction methods. Homogenization in combination with ultrasonication resulted in an enhancement in efficiency by 9.3% over homogenization alone. Similarly, maceration in combination with ultrasonication resulted in an enhancement in efficiency by 31% over maceration alone. Among all the methods employed, maceration in combination with ultrasonication resulted in the highest extraction efficiency of 77 and 93% for R-PE and R-PC, respectively followed by homogenization in combination with ultrasonication (69.6% for R-PE and 74.1% for R-PC). HPLC analysis was carried out in order to ensure that R-PE was present in the extract and remained intact even after processing. Microscopic studies indicated a clear relation between the extraction efficiency of phycobiliproteins and degree of cell disruption in a given primary extraction method. These combination methods were found to be effective for extraction of phycobiliproteins from rigid biomass of Gelidium pusillum macro-algae and can be employed for downstream processing of biomolecules also from other macro-algae.

OPTIMIZATION OF CELL DISRUPTION IN RAPHIDOCELIS SUBCAPITATA AND CHLORELLA VULGARIS FOR BIOMARKER EVALUATION

Raphidocelis subcapitata and Chlorella vulgaris are bioassay microalgae with rigid cellulosic cell wall which can hinder the release of intracellular proteins often studied as toxicity biomarkers. Since cell disruption is necessary for recovering intracellular biomolecules in these organisms, this study investigated the efficiency of ultrasonication bath; ultrasonication probe; vortexer; and bead mill in disintegrating the microalgae for anti-oxidative enzyme extraction. The extent of cell disruption was evaluated and quantified using bright field microscopy. Disrupted algae appeared as ghosts. The greatest disintegration of the microalgae (83-99.6 %) was achieved using bead mill with 0.42-0.6 mm glass beads while the other methods induced little or no disruption. The degree of cell disruption using bead mill increased with exposure time, beads-solution ratio and agitation speed while larger beads caused less disruption. Findings revealed that bead milling, with specific parameters optimized, is one of the most effective methods of disintegrating the robust algal cells.

A simple method for bakers’ yeast cell disruption using a three-phase fluidized bed equipped with an agitator

A three-phase fluidized bed equipped with a turbine agitator was utilized as a simple device for disrupting bakers’ yeast cells ( Saccharomyces cerevisiae). The degree of yeast cell disruption was evaluated based on the number of broken cells and its validity was confirmed by the total amount of crude soluble proteins released and by microscopic observation. It was found that the equipment could yield 90% of yeast cell disruption. With the presence of glass beads, the degree of cell disruption became higher as agitating speed is increased. The disruption enhancement would be attributed to the grinding effect resulting from the interaction between yeast cells and glass beads. One-thousand micrometers of glass beads yielded a higher degree of disruption than larger ones. An increase in liquid flow rate hindered the degree of disruption because of shorter contact time although the shear rates in the yeast suspension would become more rigorous.

The effects of radial shock waves on gene transfer in rabbit chondrocytes in vitro

The purpose of this study was to develop a new technique of gene transfer utilizing radial shock waves. The effects of radial shock waves on gene transfer in rabbit chondrocytes were examined by varying the parameters of exposure conditions in vitro. Chondrocytes were obtained from New Zealand white rabbits and cultured in a monolayer. A luciferase-encoding gene expression vector, or vector alone, was added to chondrocyte cell suspensions, and the cells were then exposed to radial shock waves. Parameters such as pressure amplitude, number of pulses, frequency, and DNA concentration were varied, and luciferase activity was measured 48h after transfection. Transfection efficiency of radial shock waves was compared with the FuGENE6 transfection method using a green fluorescence protein (GFP)-encoding gene vector by fluorescent-activated cell sorter (FACS) analysis. Radial shock wave exposure significantly increased luciferase activity over 140-fold as compared to the control under the optimal exposure conditions. Both pressure amplitude and number of pulses were relevant to transfection efficiency and cell viability, but frequency was not. Transfection efficiency increased in a dose-dependent manner with DNA concentration. FACS analysis showed 4.74% of GFP-encoding gene using radial shock waves. FuGENE6 transfection was almost similar in transfection efficiency to radial shock wave. In spite of certain degree of cell disruption, radial shock waves significantly augmented reporter gene transfection in rabbit chondrocytes in vitro. Radial shock waves may potentially contribute to the treatment of the cartilage morbidities by enhancing the potency of tissue healing and gene transfection of growth factors.

The disruption ofSaccharomyces cerevisiae cells and release of glucose 6-phosphate dehydrogenase (G6PDH) in a horizontal dyno bead mill operated in continuous recycling mode

Baker’s yeast was disrupted in a 1.4-L stainless steel horizontal bead mill under a continuous recycle mode using 0.3 mm diameter zirconia beads as abrasive. A single pass in continuous mode bead mill operation liberates half of the maximally released protein. The maximum total protein release can only be achieved after passaging the cells 5 times through the disruption chamber. The degree of cell disruption was increased with the increase in feeding rate, but the total protein release was highest at the middle range of feeding rate (45 L/h). The total protein release was increased with an increase in biomass concentration from 10 to 50% (w/v). However, higher heat dissipation as a result of high viscosity of concentrated biomass led to the denaturation of labile protein such as glucose 6-phosphate dehydrogenase (G6PDH). As a result the highest specific activity of G6PDH was achieved at biomass concentration of 20% (ww/v). Generally, the degree of cell disruption and total protein released were increased with an increase in impeller tip speed, but the specific activity of G6PDH was decreased substantially at higher impeller tip speed (14 m/s). Both the degree of cell disruption and total protein release increased, as the bead loading increased from 75 to 85% (v/v). Hence, in order to obtain a higher yield of labile protein such as G6PDH, the yeast cell should not be disrupted at biomass concentration and impeller tip speed higher than 20% (w/v) and 10 m/s, respectively.

Characterization and on-line monitoring of cell disruption and lysis using dielectric measurement

The dielectric measurement of Escherichia coli suspension was carried out between 0.01 and 100 MHz during cell disruption using a physical method or cell lysis induced by chemicals for the purpose of on-line monitoring of cell disruption or lysis of bacterial cells. The dielectric dispersion of relative permittivity centered at about 1.4 MHz and the dielectric parameters based on the fitting calculation well reflected the damage of these cells under physical and chemical stresses. The degree of cell disruption as determined by the dielectric parameters, the amplitude of dispersion and the conductivity, well corresponded to those obtained by other conventional kinetic analyses. This methodology can be utilized for the on-line monitoring of physical cell disruption and cell lysis induced by detergent and could provide direct information to control these processes. Based on these results, the dielectric measurement was successfully applied to monitor the stress-mediated cultivation of E. coli cells.

Cell Disruption in Broiler Breast Muscle Related to Freezing Time

SUMMARY— Cell disruption, resulting from different freezing times, was evaluated by studying the composition and amount of drip obtained from broiler breast muscles after freezing and thawing. The degree of cell disruption was estimated after measuring the amount of drip released and by total solids, nitrogen and deoxyribonucleic acid (DNA) concentration of the drip. Initial drip release was noted approximately 5% hr after the frozen meat was placed in a refrigerator at 16°C, and collections were made through the 18th hr. Degree of cell disruption was not uniformly related to changes in freezing times of 0.5 to 1,494 min. In general, increased freezing time resulted in greater cell disruption; however, several exceptions were noted. Cell disruption was relatively severe for tissues frozen in 18 to 35, 87, and 252 min, and relatively low for tissues frozen in times of 1 to 18 min, 132 to 22.5 min, and longer than 1,044 min. All frozen and thawed muscles had higher contents of total solids, nitrogen and DNA than unfrozen controls.

A CRYOSTAT METHOD FOR THE EXTRACTION OF SOLUBLE TISSUE COMPONENTS.

ESTABLISHED techniques used to release the intracellular components of tissues either into solution or suspension include homogenization1,2, mincing3, crushing4, and ultrasonic vibration5. The degree of difficulty in producing tissue homogenates varies for different tissues. In the case of cellular tissues containing little connective tissue almost complete cell disruption can be obtained with any of these techniques. Hogeboom et al.6 have shown that in unfiltered rat liver homogenates they were able to find only 7 × 106 intact cells/g liver as compared with 268 × 106 free nuclei. However, as the proportion of connective tissue increases, so does the work necessary to produce the same degree of cell disruption, and loss of labile cell contents generally follows as a result of prolonged exposure of the biological material to adverse physical conditions.