Measurement of cooling and warming rates in vitrification-based plant cryopreservation protocols.

Cryopreservation protocols include the use of additives and pretreatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro-viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (Tf ) and the glass transition (TG ) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice-derived damage. Vitrification and droplet-vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between -20°C and -120°C, ranged from ca. 5°C s(-1) to 400°C s(-1) , while warming rates spanned from ca. 2°C s(-1) to 280°C s(-1) , for the different protocols and conditions studied. A wider measuring window (0°C to -150°C) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages.

Glassy state and cryopreservation of mint shoot tips.

Vitrification refers to the physical process by which a liquid supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Thus, vitrification is an effective freeze-avoidance mechanism and living tissue cryopreservation is, in most cases, relying on it. As a glass is exceedingly viscous and stops all chemical reactions that require molecular diffusion, its formation leads to metabolic inactivity and stability over time. To investigate glassy state in cryopreserved plant material, mint shoot tips were submitted to the different stages of a frequently used cryopreservation protocol (droplet-vitrification) and evaluated for water content reduction and sucrose content, as determined by ion chromatography, frozen water fraction and glass transitions occurrence by differential scanning calorimetry, and investigated by low-temperature scanning electron microscopy, as a way to ascertain if their cellular content was vitrified. Results show how tissues at intermediate treatment steps develop ice crystals during liquid nitrogen cooling, while specimens whose treatment was completed become vitrified, with no evidence of ice formation. The agreement between calorimetric and microscopic observations was perfect. Besides finding a higher sucrose concentration in tissues at the more advanced protocol steps, this level was also higher in plants precultured at 25/-1°C than in plants cultivated at 25°C.

Conventional freezing plus high pressure-low temperature treatment: Physical properties, microbial quality and storage stability of beef meat.

Meat high-hydrostatic pressure treatment causes severe decolouration, preventing its commercialisation due to consumer rejection. Novel procedures involving product freezing plus low-temperature pressure processing are here investigated. Room temperature (20°C) pressurisation (650MPa/10min) and air blast freezing (-30°C) are compared to air blast freezing plus high pressure at subzero temperature (-35°C) in terms of drip loss, expressible moisture, shear force, colour, microbial quality and storage stability of fresh and salt-added beef samples (Longissimus dorsi muscle). The latter treatment induced solid water transitions among ice phases. Fresh beef high pressure treatment (650MPa/20°C/10min) increased significantly expressible moisture while it decreased in pressurised (650MPa/-35°C/10min) frozen beef. Salt addition reduced high pressure-induced water loss. Treatments studied did not change fresh or salt-added samples shear force. Frozen beef pressurised at low temperature showed L, a and b values after thawing close to fresh samples. However, these samples in frozen state, presented chromatic parameters similar to unfrozen beef pressurised at room temperature. Apparently, freezing protects meat against pressure colour deterioration, fresh colour being recovered after thawing. High pressure processing (20°C or -35°C) was very effective reducing aerobic total (2-log(10) cycles) and lactic acid bacteria counts (2.4-log(10) cycles), in fresh and salt-added samples. Frozen+pressurised beef stored at -18°C during 45 days recovered its original colour after thawing, similarly to just-treated samples while their counts remain below detection limits during storage.

Liquid water-ice I phase diagrams under high pressure: sodium chloride and sucrose models for food systems.

The knowledge of high pressure and low temperature phase diagrams of aqueous systems is required in fields such as food sciences, biology, cryo-microscopy and geology, to reduce processing costs, improve treatments results or advance in physical phenomena understanding. The phase transition curve between liquid water and ice I for sucrose and sodium chloride solutions has been obtained for concentrations ranging from 16% to 36% and from 1.63% to 16.09% (w/w), respectively. An accurate experimental method, based on the pressurization of an ice-solution mixture, adequate to build the entire phase transition curve at constant concentration, has been developed. Simon-like equations have been used to empirically describe the phase transition curves, so that they allow easy data interpolation.

Ice VI freezing of meat: supercooling and ultrastructural studies.

While "classical" freezing (to ice I) is disruptive to the microstructure of meat, freezing to ice VI has been found to preserve it. Ice VI freeze-substitution microscopy showed no traces of structural alteration on muscle fibres compared with the extensive damage caused by ice I freezing. The different signs of the freezing volume changes associated with these two ice phases is the most likely explanation for the above effects. Ice VI exists only at high pressure (632.4-2216 MPa) but can be formed and kept at room temperature. It was found that its nucleation requires a higher degree of supercooling than ice I freezing does, both for pure water and meat. Monitoring of the freezing process (by temperature and/or pressure measurements) is, thus, essential. The possible applications of ice VI freezing for food and other biological materials and the nucleation behaviour of this ice phase are discussed.

High CO2 atmosphere modulating the phenolic response associated with cell adhesion and hardening of Annona cherimola fruit stored at chilling temperature.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) activity, tanning ability, and polyphenols levels were measured in cherimoya (Annona cherimola Mill.) fruit treated with 20% CO(2) + 20% O(2) + 60% N(2) for 1, 3, or 6 days during chilling temperature (6 degrees C) storage. The residual effect of CO(2) after transfer to air was also studied. These observations were correlated with texture and cellular characteristics, visualized by cryo-SEM. Tanning ability and the early increase in tannin polyphenols induced by chilling temperature were reduced by CO(2) treatment. Conversely, high CO(2) atmosphere enhanced the nontannin polyphenol fraction as compared with fruit stored in air. Lignin accumulation and PAL activation observed in untreated fruit after prolonged storage at chilling temperature were prevented by high CO(2). Moreover, the restraining effect on lignification was less effective when the CO(2) treatment was prolonged for 6 days. In addition, fruits held at these conditions had greater firmness and the histological characterization of the separation between cells was similar to that in untreated fruits. We conclude that CO(2) treatment modulates the phenolic response that seems to regulate the strength of cell adhesion and so to prevent hardening caused by chilling temperature storage.

Some interrelated thermophysical properties of liquid water and ice. I. A user-friendly modeling review for food high-pressure processing.

A bibliographic search yielded a set of empirical equations that constitute an easy method for the calculation of some thermophysical properties of both liquid water and ice I, properties that are involved in the modeling of thermal processes in the high-pressure domain, as required in the design of new high-pressure food processes. These properties, closely interrelated in their physical derivation and experimental measurement, are specific volume, specific isobaric heat capacity, thermal expansion coefficient, and isothermal compressibility coefficient. Where no single equation was found, an alternative method for calculation is proposed. Keeping in mind the intended applications and considering the availability of both experimental data and empirical equations, the limits for the set of equations where set in -40 to 120 degrees C and 0 to 500 MPa for liquid water and -30 to 0 degrees C and 0 to 210 MPa for ice I. The equations and methods selected for each property are described and their results analyzed. Their good agreement with many existing experimental data is discussed. In addition, the routines implemented for the calculation of these properties after the described equations are made available in the public domain.

A model for real thermal control in high-pressure treatment of foods.

A model for the simulation of thermal exchanges in a complete high-pressure equipment was developed. Good agreement between simulated and experimental time-temperature profiles was found during different processes of pressurization and depressurization. The model allows study of the effect of different variables to improve thermal control in the treatments performed. This work involved an important advance in optimization and regulation of high-pressure processes in the food industry.