Interaction of pressure, time and temperature of pressurization on viability loss of Listeria innocua

In this study, the combined effectiveness of pressures of 137.9–344.7MPa, temperatures of 25–50°C and exposure times of 5–15min on Listeria innocua viability in peptone solution is examined. The results showed that under the study conditions only the combination of 344.7MPa, 50°C and 9.1min can reduce the viability of this species by 7 logs, with a z value of 173.1MPa.     

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel,fluid or liquid-ordered phase,determined using AFM force spectroscopy

The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20 °C and 50 °C using atomic force microscopy (AFM) and force spectroscopy. At 20 °C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10–20 MPa and 4–6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0–5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50 °C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties.     

Physiological manipulation and formulation of the biocontrol yeast Pichia anomala for control of Penicillium verrucosum and ochratoxin A contamination of moist grain

The major hurdle in the production of commercial biocontrol agents (BCAs) has been the lack of production of appropriate formulations. Of particular importance is the conservation of viability and ecological competence after application. With this in mind studies were conducted to develop formulations of P. anomala which would have these attributes. Cells were grown in molasses-based medium modified with proline to different water availability levels (0.98 and 0.96) which significantly increased (up to 50%) the content of trehalose and arabitol in the yeast cells during liquid broth fermentation. The use of isotonic solutions for harvesting the yeast cells further increased the endogenous content of these compatible solutes as well as glycerol. Fluidised bed drying of cells at 30–80°C was carried out for 10 and 20 min and showed that viability was significantly decreased at 70–80°C. A temperature of 50°C for 20 min was found to be best for viability (70%) and moisture content of <10%. Several additives for conservation of viability showed that cotton seed flour+skimmed milk was the best treatment when dried at 50°C. The biocontrol efficacy of formulated P. anomala cells was tested in laboratory scale studies and this showed that they inhibited growth of Penicillium verrucosum and reduce ochratoxin A production in moist wheat grain under some combinations of water availability. Physiologically modified formulated yeast cells with increased levels of trehalose and arabitol gave similar efficacy as fresh cells. This suggests that ecophysiological manipulation of such BCAs can result in improved ecological competence of such formulations and effective biocontrol.     

Effect of freezing and freeze-drying on the viability and storage of Lilium longiflorum L. and Zea mays L. pollen

The freeze-preservation of pollen is dependent on the interaction of several factors such as freezing rate, thawing rate, freeze-drying temperature and duration, storage temperature and environment and rehydration rates. Changes in any of these variables affects the others directly or indirectly.Rapid freezing of pollen at rates of approximately 200 °C/min maintains the highest degree of viable pollen in combination with rapid thawing rates of 218 °C/min. Rapid cooling and slow rewarming resulted in a substantial loss of pollen viability. This might indicate that intracellular ice crystals formed during rapid cooling perhaps grow into larger ice masses during slow rewarming or storage at temperatures above ?50 °C.The germinability of pollen freeze-dried at temperatures below ?50 °C was also prolonged over that of the controls. Germination values for unfrozen pollen stored for 30 days at 0–5 °C averaged 50% for lily and 20% for corn. Freeze-dried pollen stored for 30 days at the same temperature yielded considerably higher viability percentages for both lily and corn pollen. Drying time is an important factor, perhaps indicating that residual moisture is critical. Freeze-dried pollen can be stored at higher temperatures than frozen and control pollen. Freeze-dried material stored for five months at 0–5 °C, upon slow rehydration yielded intact grains which has average germination percentages of 25 for lily and 15 for corn. The same pollen upon rapid rehydration showed rupturing of 20–40% of the cells and practically no germination.     

Effects of different heat–moisture treatments on the physicochemical properties of brown rice flour

We evaluated the effect of heat–moisture treatment (HMT) on the main chemical components, physical properties, and enzyme activities of two types of brown rice flour: high-amylose Koshinokaori and normal-quality Koshiibuki. Five different HMTs using brown rice (moisture content was 12.0%) were assessed: 0.1 MPa/120 °C for 5 or 10 min, 0.2 MPa/134 °C for 5 or 10 min and 0.3 MPa/144 °C for 10 min. HMT, decreased the α-amylase and lipase activities, and fat acidity, and slightly increased the dietary fiber and resistant starch levels. After 2 months’ storage at 35 °C, rice samples that were treated with 0.2 MPa/134 °C or 0.3 MPa/144 °C for 10 min had a lower fat acidity than untreated samples, which would be useful for long-term storage and export of rice flour. And HMT exhibited inhibition of retrogradation in the pasting and physical properties, which is profitable to promote the qualities of the rice products.     

Fertilizability of cryopreserved zona-free hamster ova

Mature unfertilized ova from superovulated hamsters were freed from all investments and frozen at ?50°C. They were cooled at about 1°C/min to 0°C then at 0.8° to 0.6°C/min to ?50°C. At 0°C, dimethyl sulfoxide was added to a final concentration of 1.25 M. The ova were stored at ?50°C for up to four months. Thawing was performed at 2–4°C/min and followed by several washes with insemination medium. Approximately 90% of the ova were normal in appearance after thawing. The frozen and thawed ova with normal appearance could be penetrated by hamster or human spermatozoa at a rate comparable to unfrozen controls. The ability of hamster ova to tolerate storage at a relatively convenient temperature (?50°C) for long periods (tested for up to four months) makes possible their shipment at low cost to institutions lacking this resource. There they can be used for basic biological studies of sperm–egg interaction or in the clinical assessment of human sperm quality.     

Cryopreservation of cell line Paesun by a rapid cooling

The purpose of the present study was to clarify the possibility of a rapid cryopreservation for cell line Paesun by cooling in the range of 30–40 °C/min to vapor phase of −120 ∼-140 °C before immersion into liquid phase of liquid nitrogen using 10% Me₂SO. After thawing, these cells were examined with assaying viability by trypan blue exclusion staining and survival by cloning in monolayer; the percentages of cell and colony recovery obtained in rapid cooling had a tendency to be lower than that by slow cooling of 1 °C/min but there were no significant differences between them. In addition, post-thaw cells were examined by assaying proliferation and susceptibility to virus lines; there were no significant differences between before and after cryopreservation. In conclusion, these findings indicate that Paesun can be successfully cryopreserved by the rapid cooling rate of 30 °C–40 °C/min.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Some factors affecting the viability of mouse and cattle embryos frozen to −40°C before transfer to liquid nitrogen

A total of 1161 8- to 16-cell mouse embryos and 31 cattle early morulae and late blastocysts were frozen to ?40°C before transfer to liquid nitrogen. After thawing, mouse embryo viability was determined by in vitro development to the blastocyst stage and cattle embryo viability by both in vivo and in vitro development.Using glycerol as the cryoprotective agent, 88% of the mouse embryos developed to the blastocyst stage: thawing at 45 and 360° C/min gave the best results (88.8 and 84.8%, respectively). In another test with holding times at ?40°C of up to 60 min, about 70% of embryos developed to blastocysts with holding time 30–60 min.In cattle, 11 embryos frozen in DMSO and thawed at 360°C/min were transplanted to eight recipients. Four pregnancies (six fetuses) resulted. Thawing rates of 200 and 360°C/min resulted in the best in vitro development of cattle embryos.     

The stability of almond β-glucosidase during combined high pressure–thermal processing: a kinetic study

The thermal and the combined high pressure–thermal inactivation kinetics of almond β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) were investigated at pressures from 0.1 to 600 MPa and temperatures ranging from 30 to 80 °C. Thermal treatments at temperatures higher than 50 °C resulted in significant inactivation with complete inactivation after 2 min of treatment at 80 °C. Both the thermal and high pressure inactivation kinetics were described well by first-order model. Application of pressure increased the inactivation kinetics of the enzyme except at moderate temperatures (50 to 70 °C) and pressures between 0.1 and 100 MPa where slight pressure stabilisation of the enzyme against thermal denaturation was observed. The activation energy for the inactivation of the enzyme at atmospheric pressure was estimated to be 216.2?±?8.6 kJ/mol decreasing to 55.2?±?3.9 kJ/mol at 600 MPa. The activation volumes were negative at all temperature conditions excluding the temperature–pressure range where slight pressure stabilisation was observed. The values of the activation volumes were estimated to be ?29.6?±?0.6, ?29.8?±?1.7, ?20.6?±?3.2, ?41.2?±?4.8, ?36.5?±?1.8, ?39.6?±?4.3, ?31.0?±?4.5 and ?33.8?±?3.9 cm³/mol at 30, 35, 40, 45, 50, 60, 65 and 70 °C, respectively, with no clear trend with temperature. The pressure–temperature dependence of the inactivation rate constants was well described by an empirical third-order polynomial model.     

Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus

We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus. Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected. Growth at 60 MPa was slower. The optimal growth temperature shifted from 85° C at 30 MPa to 90–95° C at 45 MPa. Cell viability during the stationary phase was also enhanced under high pressure. A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures. The viability of cells during starvation, at high temperature (90, 95° C), and at low temperature (10° C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure. These results show that the extremely thermophilic archaeon T. peptonophilus is a barophile. Received: 21 October 1996 / Accepted: 5 February 1997     

Viability of plum ovules at different temperatures

The viability of ovules was studied in five plum cultivars under laboratory conditions at four constant temperatures: 5°C, 10°C, 15°C and 20°C and under field conditions over two years. During 10 days from the onset of full bloom, ovule viability in cvs ‘?a?anska Rana’, ‘?a?anska Najbolja’ and ‘?a?anska Lepotica’ was between 80–100 % at the temperatures of 5°C, 10°C and 15°C, in both years. In the same period, ovule viability in cvs ‘Wangenheims Frühzwetsche’ and ‘Po?ega?a’ was lower, but never below 50%. At the constant temperature of 20°C, all plum cultivars showed a decline in longevity of ovule viability, which was pronounced in cv. ‘?a?anska Rana’. During the 10 days from the onset of full bloom, ovule viability in all five plum cultivars under field conditions showed a high viability, which approximated to the ovule viability of the cultivars at the constant temperatures of 5°C, 10°C and 15°C, in both years. Determination of the longevity of ovule viability in the mentioned plum cultivars is of great importance due to its effect on the effective pollination period and fertilisation success. This paper deals in detail with the interrelations between the temperature effects on ovule viability, pollen tube growth and fertilisation, as well as on fruit setting.     

Critical temperature for skin necrosis in experimental cryosurgery

To investigate the minimal lethal freezing temperature required to produce skin necrosis in dogs, multiple skin sites were frozen with cryosurgical equipment. Tissue temperatures were recorded from thermocouple sites placed at diverse distances, usually 5 mm from the edge of the freezing probe. In single freezing cycles of about 3 min duration, tissue temperatures in the range of 0 to ?60 °C were produced. Punch biopsies of the skin at the thermocouple sites 3 days after freezing injury provided tissues for estimation of viability by histologic examination.The histologic findings permitted classification of the biopsy tissue into three groups, that is, viable, borderline, or necrotic. When classified as borderline, the division between the necrotic and viable tissue was evident on the histologic slide. The viable specimens were scattered through the 0 to ?35 °C range. All specimens frozen to ?10 °C or warmer were viable. In biopsies classified as borderline, the range of viability extended from ?11 ° to ?50 °C. The necrotic biopsies covered a range of ?14 ° to ?50 °C. Cell death was certain at temperatures colder than ?50 °C. The data showed cryosurgical freezing conditions produced a range of temperatures in which viability or death of tissue may occur and that the ranges of viability and necrosis overlapped to a great extent.The wide range of temperatures at which cells were viable shows the need to achieve tissue temperatures in the range of ?50 °C in the cryosurgical treatment of cancer.     

The Impact of Temperature Stress on DNA and RNA Synthesis in Potentially Toxic Dinoflagellates <Emphasis Type="Italic">Prorocentrum minimum</Emphasis>

Biomarkers of temperature stress were studied as major characteristics crucial for the understanding complex processes that underlie the response of marine planktonic microorganisms to environmental factors and their sublethal effects. Using the potentially toxic dinoflagellates Prorocentrum minimum as a model object, the impact of temperature stress on viability, cell cycle, RNA synthesis and DNA replication in these protists was evaluated. It was shown by flow cytometry that stress evoked by a temperature increase from 25°C (control) to 37 or 42°C during 15 to 60 min did not cause any considerable alterations in the cell cycle, while cell death rate increased from ≤ 1% (control) to 2–12% at 37°C and 4–22% at 42°C. Along with a relatively low cell death rate, following a temperature increase to 37 and/or 42°C, P. minimum displayed the ability to boost the synthesis of DNA (1.7–1.9 and 1.2–1.6 times, respectively) and especially RNA (2.5–3.1 and 1.7–2.8 times, respectively) during the first 15–30 min after stress. At certain stages of the life cycle, this effect can be critical for maintaining the viability and normal development of the P. minimum population. The obtained results demonstrate that a significantly elevated synthesis of nucleic acids can serve as an indicator (biomarker) of sublethal environmental stress.     

Temperature, soluble solids and pH effect on Alicyclobacillus acidoterrestris viability in lemon juice concentrate

Alicyclobacillus acidoterrestris is a thermoacidophilic, non-pathogenic, spore-forming bacterium detected in spoiled commercial pasteurized fruit juice. Apple, white grape and tomato are particularly susceptible. A. acidoterrestris spores are resistant to lemon juice pasteurization (2 min at 82°C), and they can germinate and grow causing spoilage. This contamination is characterized by a medicinal or disinfectant smell attributed to guaiacol (o-dihydroxybenzene) production and other taint chemicals. The aim of this work was to study the influence of temperature (82, 86, 92 and 95 °C), total soluble solids (SS) (6.20, 9.8, 50 and 68°Brix) and pH (2.28, 2.45, 2.80, 3.25, 3.5) on decimal reduction time (D) of the A. acidoterrestris in clarified and non-clarified concentrated lemon juice. Once D-value was determined, the resistance of A. acidoterrestris at the assayed temperatures was confirmed. SS and pH influence spore viability, because spore resistance increases with higher SS (50°Brix 22 min 82 °C–68°Brix 28 min 82 °C) and pH values (pH 2.28, 17 min–pH 4.00, 22 min). Bacterial growth was lower in clarified lemon juice, 26 min at 82 °C, than in non-clarified lemon juice, 51 min at 82 °C. Temperature was the parameter that had the greatest influence on the D value.     

The developmental potential of frozen-thawed hamster preimplantation embryos following embryo transfer: Viability of slowly frozen embryos following slow and rapid thawing

Hamster preimplantation embryos were slowly frozen (0.33°C/min) and seeded above 10°C in TC-199 containing 1.5 M-DMSO. These embryos were thawed either slowly (1.5°C/min) or rapidly (90°C/min). The thawed embryos were examined by morphology, trypan blue exclusion and viability after embryo transfer. Slow thawing gave significantly higher viability compared to rapid thawing. The early preimplantation embryos demonstrate higher sensitivity to freezing. The three tests of viability (morphology, trypan blue exclusion and embryo transfer) were found to be positively correlated.     

Effect of hot-water treatments in vitro on conidial germination and mycelial growth of grapevine trunk pathogens

In this study, the sensitivity of Cadophora luteo-olivacea, Cylindrocarpon liriodendri, Cn. macrodidymum and eight species of the genus Phaeoacremonium to hot-water treatments (HWTs) in vitro was evaluated. Conidial suspensions and plugs of agar with mycelia were placed in Eppendorf vials and incubated for 30, 45 or 60 min in a hot-water bath at 41, 42, 43, 44, 45, 46, 47, 48 or 49°C for Cylindrocarpon spp. and at 49, 50, 51, 52, 53, 54 or 55°C for Ca. luteo-olivacea and Phaeoacremonium spp. In general, conidial germination and the colony growth rate of all pathogens decreased with increased temperature and time combinations. Cylindrocarpon spp. were more sensitive than Ca. luteo-olivacea and Phaeoacremonium spp. to HWT temperatures. Conidial germination of Ca. luteo-olivacea was inhibited by treatments above 51°C–30 min, while treatments up to 54°C–60 min were necessary to inhibit the mycelial growth. For Cylindrocarpon spp., conidial germination was inhibited by treatments above 45°C–45 min, while treatments above 48°C–45 min were necessary to inhibit the mycelial growth. Regarding Phaeoacremonium spp., treatments up to 54°C–60 min were necessary to completely inhibit both conidial germination and mycelial growth. These results suggest that current HWT protocols at 50°C for 30 min may be sufficient to control Cylindrocarpon spp. However, it would be necessary to develop HWT using higher temperatures to reduce the incidence of Ca. luteo-olivacea and Phaeoacremonium spp. infections.     

Short‐term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short‐term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30–37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30‐min preincubation of isolated mitochondria at 25–40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short‐term high temperature events associated with global warming.     

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42°–45.5°C and for 10–180 min) was examined. Synthesis of 70 kDa hsp (hsp 70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42°C-10 min (236.6 ± 71.4; P < 0.05) and 43°C-30 min (276.8 ± 89.4; P < 0.005) compared to control (173.9 ± 53.9). The 42°C-180 min group (158.0 ± 27.1 μm) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 ± 47.3 μm), while heat stress with 43°C for ≧60 min, 44°–44.5°C for ≧30 min, or 45°-45.5°C for ≧10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42°C-180 min, 43°C-10 min, 43°C-30 min, 44°C-10 min, or 45°C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42°C for 180 min, 43°C for 30 min, 44°C for 10 min, and 45°C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear. © 1996 Wiley-Liss, Inc.     

Improving of Catalase Stability Properties by Encapsulation in Alginate/Fe3O4 Magnetic Composite Beads for Enzymatic Removal of H2O2

The aim of this study was enhancing of stability properties of catalase enzyme by encapsulation in alginate/nanomagnetic beads. Amounts of carrier (10–100 mg) and enzyme concentrations (0.25–1.5 mg/mL) were analyzed to optimize immobilization conditions. Also, the optimum temperature (25–50°C), optimum pH (3.0–8.0), kinetic parameters, thermal stability (20–70°C), pH stability (4.0–9.0) operational stability (0–390 min), and reusability were investigated for characterization of the immobilized catalase system. The optimum pH levels of both free and immobilized catalase were 7.0. At the thermal stability studies, the magnetic catalase beads protected 90% activity, while free catalase maintained only 10% activity at 70°C. The thermal profile of magnetic catalase beads was spread over a large area. Similarly, this system indicated the improving of the pH stability. The reusability, which is especially important for industrial applications, was also determined. Thus, the activity analysis was done 50 times in succession. Catalase encapsulated magnetic alginate beads protected 83% activity after 50 cycles.