Controlled Ice Nucleation Research Articles

Investigation of Two Different Pressure-Based Controlled Ice Nucleation Techniques in Freeze-Drying: The Integral Role of Shelf Temperature After Nucleation in Process Performance and Product Quality

The purpose of this study was to investigate the impact of shelf temperature modifications during application of controlled ice nucleation techniques on process data and critical product quality attributes for a challenging, high-concentration and high-fill volume amorphous model system. Different freezing programs were applied and compared for the mechanistically different depressurization and vacuum-induced surface freezing techniques. Critical process data, such as product temperature and drying time, were analyzed. The final products were characterized with a focus on product morphology, residual moisture, reconstitution time and stability. The shelf temperature directly after primary nucleation showed a major influence on process performance and product quality attributes, with an isothermal hold step at an intermediate temperature leading to optimal results in terms of homogeneity and reduction of product temperatures and drying time for the model system used. The different controlled ice nucleation techniques led to significantly different results in terms of product morphology and process data, showing that the two mechanistically different controlled nucleation techniques are not interchangeable.

Dynamical in-situ observation of the lyophilization and vacuum-drying processes of a model biopharmaceutical system by an environmental scanning electron microscope.

The paper discusses the real-time monitoring of the changing sample morphology during the entire lyophilization (freeze-drying) and vacuum-drying processes of model biopharmaceutical solutions by using an environmental scanning electron microscope (ESEM); the device's micromanipulators were used to study the interior of the samples in-situ without exposing the samples to atmospheric water vapor. The individual collapse temperatures (Tc) of the formulations, pure bovine serum albumin (BSA) and BSA/sucrose mixtures, ranged from -5 to -29°C. We evaluated the impact of the freezing method (spontaneous freezing, controlled ice nucleation, and spray freezing) on the morphologies of the lyophiles at the constant drying temperature of -20°C. The formulations with Tc above -20°C resulted in the lyophiles' morphologies significantly dependent on the freezing method. We interpret the observations as an interplay of the freezing rates and directionalities, both of which markedly influence the morphologies of the frozen formulations, and, subsequently, the drying process and the mechanical stability of the freeze-dried cake. The formulation with Tc below -20°C yielded a collapsed cake with features independent of the freezing method. The vacuum-drying produced a material with a smooth and pore-free surface, where deep cracks developed at the end of the process.

Stability of Freeze-Dried Protein Formulations: Contributions of Ice Nucleation Temperature and Residence Time in the Freeze-Concentrate

Controlling ice nucleation, at a fixed higher temperature, results in larger ice crystals, which can reduce the ice/freeze-concentrate interface area where proteins can adsorb and partially unfold. Moreover, limited work has been done to address any effects on short-term stability due to a slow ramp or long isothermal hold after the ice nucleation step. The objective was to evaluate the effect of the ice nucleation temperature and residence time in the freeze-concentrate on in-process or storage stability of representative proteins, human IgG, and recombinant human serum albumin. The results suggest a higher ice nucleation temperature can minimize aggregation of protein pharmaceuticals, which are labile at ice/aqueous interface. Apart from the ice nucleation step, the present study identified the residence time in the freeze-concentrate as the critical factor that influences protein stability post ice nucleation. At a temperature where enough mobility exists (i.e., above Tg’ of the formulation), the long residence time in the freeze-concentrate can result in significant protein aggregation during the process. In addition to stability, the findings revealed that not only the ice nucleation temperature but also the thermal history of the formulation post ice nucleation defines the surface area of ice and the porous structure of the freeze-dried cake.

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below −20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.

Control of Ice Nucleation for Subzero Food Preservation

Freezing processes have long been employed for the preservation of foods, providing minimum nutrition loss with a long shelf life period. Freezing plays an essential role in ensuring the safety of food products in all regions of the world. Nonetheless, slow freezing rates and frequently freezing/thawing lead to permanent physicochemical changes, damage of structure, degradation of nutrition values, and color changes through the formation of large ice crystals in food matrix during the cold storage. The size of ice crystals is highly related to the duration of phase transition and degree of supercooling. This paper reviews that the degree of supercooling and nucleation temperature can be controlled by positive or negative pressure and vibration of the dipole and dipole rotation water molecule techniques. Controlling nucleation temperature and suppression of ice crystals in the food matrix could not be achieved by current freezing methods such as air blast, contact, and immersion freezing in the food industry. These present freezing methods are especially focused on increasing the heat transfer rate in foods. Rapid freezing technology may depend on the size and shape of food. However, the size of ice crystals and the suppression of nucleation could be achieved by alternative freezing technologies to overcome the drawbacks of current freezing technologies in the food industry. Conventional freezing technologies can be replaced by emerging freezing techniques, ultrasound irradiation, high pressure, electric field, magnetic field, and microwave-assisted freezing to control the properties of the nucleation and degree of supercooling in the food industry.

Reconstitution of Highly Concentrated Lyophilized Proteins: Part 1 Amorphous Formulations

Long reconstitution times before patient administration remain an undesirable quality attribute for high concentration lyophilized protein formulations. In this study, 3 approaches were developed to study reconstitution behavior of lyophilized, amorphous cakes of a highly concentrated monoclonal antibody (mAb) by exploring their wetting, disintegration, and hydration behavior. As the mAb concentration increased from 0 to 83 mg/mL, reconstitution times were longer with poorer wetting, slower hydration, and disintegration rates. Furthermore, the effect of controlling ice nucleation temperature at −5 and −10°C during freezing followed by either conservative or aggressive drying conditions on the reconstitution times was explored in formulations containing 40 and 83 mg/mL mAb. Although no effect of either of the 2 processing conditions was noted at 40 mg/mL, aggressive drying led to faster reconstitution at both the nucleation temperatures with 83 mg/mL mAb. The present study combined with literature data suggests that below a protein-to-sugar ratio of 1, reconstitution was complete within 1 min, and when the ratio was greater than 1, the reconstitution times increased nonlinearly. Disintegration and hydration were determined to be the key mechanisms contributing to the complete reconstitution of the lyophilized, amorphous cakes of the highly concentrated mAb in vials.

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry (ssHDX-MS).

Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) was evaluated as an analytical method to rapidly screen and select an optimal lyophilized fragment antigen binding protein (Fab) formulation and the optimal lyophilization cycle. ssHDX-MS in lyophilized Fab formulations, varying in stabilizer type and stabilizer/protein ratio, was conducted under controlled humidity and temperature. The extent of deuterium incorporation was measured using mass spectrometry and correlated with solid-state stress degradation at 50 °C as measured by size exclusion chromatography (SEC) and ion-exchange chromatography (IEC). ssHDX-MS was also used to evaluate the impact of three different types of lyophilization processing on storage stability: controlled ice nucleation (CN), uncontrolled ice nucleation (UCN), and annealing (AN). The extent of deuterium incorporation for different Fab formulations agreed with the order of solid-state stress degradation, with formulations having lower deuterium incorporation showing lower stress-induced degradation (aggregation and charge modifications). For lyophilization processing, no significant effect of ice nucleation was observed in either solid-state stress degradation or in the extent of deuterium incorporation for high concentration Fab formulations (25 mg/mL). In contrast, for low concentration Fab formulations (2.5 mg/mL), solid-state stability from different lyophilization processes correlated with the extent of deuterium incorporation. The order of solid-state degradation (AN < CN < UCN) was the same as the extent of deuterium incorporation on ssHDX-MS (AN < CN < UCN). The extent of deuterium incorporation on ssHDX-MS correlated well with the solid-state stress degradation for different Fab formulations and lyophilization processing methods. Thus, ssHDX-MS can be used to rapidly screen and optimize the formulation and lyophilization process for a lyophilized Fab, reducing the need for time-consuming stress degradation studies.

Frost Survival Mechanism of Vegetative Buds in Temperate Trees: Deep Supercooling and Extraorgan Freezing vs. Ice Tolerance.

In temperate climates, overwintering buds of trees are often less cold hardy than adjoining stem tissues or evergreen leaves. However, data are scarce regarding the freezing resistance (FR) of buds and the underlying functional frost survival mechanism that in case of supercooling can restrict the geographic distribution. Twigs of 37 temperate woody species were sampled in midwinter 2016 in the Austrian Inn valley. After assessment of FR, infrared-video-thermography and cryo-microscopy were used to study the freezing pattern in and around overwintering vegetative buds. Only in four species, after controlled ice nucleation in the stem (−1.6 ± 0.9°C) ice was observed to immediately invade the bud. These buds tolerated extracellular ice and were the most freezing resistant (−61.8°C mean LT50). In all other species (33), the buds remained supercooled and free of ice, despite a frozen stem. A structural ice barrier prevents ice penetration. Extraorgan ice masses grew in the stem and scales but in 50% of the species between premature supercooled leaves. Two types of supercooled buds were observed: in temporary supercooling buds (14 species) ice spontaneously nucleated at −20.5 ± 4,6°C. This freezing process appeared to be intracellular as it matched the bud killing temperature (−22.8°C mean LT50). This response rendered temporarily supercooled buds as least cold hardy. In 19 species, the buds remained persistently supercooled down to below the killing temperature without indication for the cause of damage. Although having a moderate midwinter FR of −31.6°C (LT50), some species within this group attained a FR similar to ice tolerant buds. The present study represents the first comprehensive overview of frost survival mechanisms of vegetative buds of temperate trees. Except for four species that were ice tolerant, the majority of buds survive in a supercooled state, remaining free of ice. In 50% of species, extraorgan ice masses harmlessly grew between premature supercooled leaves. Despite exposure to the same environmental demand, midwinter FR of buds varied intra-specifically between −17.0 and −90.0°C. Particularly, species, whose buds are killed after temporary supercooling, have a lower maximum FR, which limits their geographic distribution.

New Insights in Potato Leaf Freezing by Infrared Thermography.

Infrared thermography has been widely used to study freezing processes in freezing resistant plants but hardly in freezing susceptible species. Solanum tuberosum leaves get frost killed at −3 °C and are unable to frost harden. The basic nature of frost injury to potato leaves is not clear. By employment of infrared differential thermal analysis (IDTA) in combination with viability assessment, we aimed to clarify the mechanistic relationship between ice formation and frost injury. During controlled freezing of potato leaves two distinct freezing events were detected by IDTA. During the first freezing event, the ice wave propagated via the xylem and spread out within 60 s throughout the whole leaf. When leaves were rewarmed after this freezing event, they did not show any frost injury symptoms. We suggest that this non-lethal first ice wave is restricted to the extracellular space. When leaves remained exposed after this exotherm, a second freezing event with a diffuse freezing pattern without a distinct starting point was recorded. When thawed after this second freezing event, leaves always showed frost damage suggesting intracellular freezing. The freezing behavior of potato leaves and its relation to frost damage corroborates that control of ice nucleation is a key for frost protection.

Unexplored benefits of controlled ice nucleation: Lyophilization of a highly concentrated monoclonal antibody solution

The study explores new benefits of controlled ice nucleation during lyophilization. A highly concentrated monoclonal antibody product was lyophilized using conventional (uncontrolled) and controlled ice nucleation freezing and the processes were compared for their impact on process performance, reconstitution time, and product quality attributes. Glass fogging was observed during the conventional ice nucleation freezing step and is investigated. A mechanical stress study was also conducted on the lyophilized product manufactured using uncontrolled and controlled ice nucleation to measure and compare propensity toward aggregation upon physical stresses associated with shipping and handling. Reduction in primary drying time and reconstitution time was achieved with controlled ice nucleation. In contrast to significant fogging observed for uncontrolled nucleation, glass fogging was absent in vials subjected to controlled ice nucleation. Uncontrolled nucleation freezing produced higher number of particles when subjected to mechanical stress prior to reconstitution compared to controlled nucleation lyophilized samples. The study confirms published benefits of controlled ice nucleation (e.g. reduction in primary drying time). The manuscript reports for the first time the advantage that controlled ice nucleation may offer in regards to glass fogging during lyophilization. The reduced number of particles in the controlled nucleated samples exposed to mechanical stress suggest that frictional forces play an important role in governing the stability of protein in the lyophilized state.

Ice adhesion mechanism on lubricant-impregnated surfaces using molecular dynamics simulations

ABSTRACTIce formation causes numerous problems in many industrial fields as well as in our daily life. The control of ice nucleation and rational design of anti-icing surface with low ice adhesion are desirable in various industries such as aircraft, power line, ships, building, and cryopreservation. However, despite considerable attention in the development of ice or water-repellent surfaces, it is still challenging to design icephobic or anti-icing surfaces with high resistance to icing. In this study, coarse-grained molecular dynamics simulation is utilised to investigate the ice adhesion mechanism on lubricant-infused nanotextured surfaces. Using steered molecular dynamics simulation, we find that the adhesion strength of ice on nanotextured surfaces impregnated with lubricant films to be higher compared to that on textured surfaces in presence excess lubricant films. We illustrate that the ice adhesion strength depends on the texture density and the ice adhesion strength increases with nanoposts density. Lubricant-impregnated surfaces (LISs) with higher posts density exhibit greater adhesive interaction energy due to the large contact area between the icecube and the textured surface. This systematic study enhances our understanding of ice adhesion mechanism on LISs which can apply for designing novel anti-icing surfaces with extremely weak ice adhesion strength.

Ice induction in DSC experiments with Snomax®

For differential scanning calorimetry (DSC) analysis, controlling the degree of supercooling at which crystallization begins may be required for several studies, experiments, and applications. In this paper, the use of Snomax, an ice nucleating agent (INA), was evaluated to create ice at a desirable temperature range in a DSC aluminum sample pan. The effect of Snomax on the nucleation temperature (Tn) was studied in pure water. Best practices and methods are described in terms of the Tn dependence on three experimental parameters: (i) the Snomax concentration that controls the Tn value for different groups according to the three classes of the Pseudomonas syringae protein aggregates (from which Snomax originates); (ii) the sample volume that affects the presence probabilities of the different INA subpopulations in the solution and that could also favor their deterioration; and (iii) the cooling rate that does not seem to further affect the Tn value. There is no-evidence of time dependence of the nucleation process promoted by Snomax. The presence of artifacts or disturbances introduced by the addition of Snomax into the solution was evaluated. No major disturbances of the thermodynamic characteristics of these solutions were observed with the addition of Snomax below 103 mg L−1 concentration. This underscores the possible use of Snomax for controlling ice nucleation during DSC experiments.

A Comparison of Controlled Ice Nucleation Techniques for Freeze-Drying of a Therapeutic Antibody

The aim of this study was to investigate if mechanistically different controlled ice nucleation techniques in freeze-drying are comparable to each other with respect to drying process performance and product quality attributes. Therefore, we studied 3 different model formulations including amorphous (sucrose, trehalose) and semi-crystalline (mannitol:sucrose 4:1) solids containing a monoclonal antibody IgG1 (5 g/L) processed either by application of ice fog or depressurization technique setting an ice nucleation temperature of −5°C. Subsequently, the same freeze-drying protocol on identical machinery was applied. The results showed that the techniques are comparable with respect to the thermal history of product temperature sensors and primary drying time, solid state- and protein-related product quality attributes. All analytics comprising Karl Fischer titration, X-ray powder diffraction and Brunauer–Emmet–Teller as well as high-performance size exclusion chromatography, turbidity and subvisible particle counting using flow-imaging microscopy exhibited similarity and comparability among the controlled nucleation protocols.

Determination of the dried product resistance variability and its influence on the product temperature in pharmaceutical freeze-drying

During the primary drying step of the freeze-drying process, mass transfer resistance strongly affects the product temperature, and consequently the final product quality. The main objective of this study was to evaluate the variability of the mass transfer resistance resulting from the dried product layer (Rp) in a manufacturing batch of vials, and its potential effect on the product temperature, from data obtained in a pilot scale freeze-dryer. Sublimation experiments were run at −25 °C and 10 Pa using two different freezing protocols: with spontaneous or controlled ice nucleation. Five repetitions of each condition were performed. Global (pressure rise test) and local (gravimetric) methods were applied as complementary approaches to estimate Rp. The global method allowed to assess variability of the evolution of Rp with the dried layer thickness between different experiments whereas the local method informed about Rp variability at a fixed time within the vial batch. A product temperature variability of approximately ±4.4 °C was defined for a product dried layer thickness of 5 mm. The present approach can be used to estimate the risk of failure of the process due to mass transfer variability when designing freeze-drying cycle.

Bioinspired Materials for Controlling Ice Nucleation, Growth, and Recrystallization.

Ice formation, mainly consisting of ice nucleation, ice growth, and ice recrystallization, is ubiquitous and crucial in wide-ranging fields from cryobiology to atmospheric physics. Despite active research for more than a century, the mechanism of ice formation is still far from satisfactory. Meanwhile, nature has unique ways of controlling ice formation and can provide resourceful avenues to unravel the mechanism of ice formation. For instance, antifreeze proteins (AFPs) protect living organisms from freezing damage via controlling ice formation, for example, tuning ice nucleation, shaping ice crystals, and inhibiting ice growth and recrystallization. In addition, AFP mimics can have applications in cryopreservation of cells, tissues, and organs, food storage, and anti-icing materials. Therefore, continuous efforts have been made to understand the mechanism of AFPs and design AFP inspired materials. In this Account, we first review our recent research progress in understanding the mechanism of AFPs in controlling ice formation. A Janus effect of AFPs on ice nucleation was discovered, which was achieved via selectively tethering the ice-binding face (IBF) or the non-ice-binding face (NIBF) of AFPs to solid surfaces and investigating specifically the effect of the other face on ice nucleation. Through molecular dynamics (MD) simulation analysis, we observed ordered hexagonal ice-like water structure atop the IBF and disordered water structure atop the NIBF. Therefore, we conclude that the interfacial water plays a critical role in controlling ice formation. Next, we discuss the design and fabrication of AFP mimics with capabilities in tuning ice nucleation and controlling ice shape and growth, as well as inhibiting ice recrystallization. For example, we tuned ice nucleation via modifying solid surfaces with supercharged unfolded polypeptides (SUPs) and polyelectrolyte brushes (PBs) with different counterions. We found graphene oxide (GO) and oxidized quasi-carbon nitride quantum dots (OQCNs) had profound effects in controlling ice shape and inhibiting ice growth. We also studied the ion-specific effect on ice recrystallization inhibition (IRI) with a large variety of anions and cations. All functionalities are achieved by tuning the properties of interfacial water on these materials, which reinforces the importance of the interfacial water in controlling ice formation. Finally, we review the development of novel application-oriented materials emerging from our enhanced understanding of ice formation, for example, ultralow ice adhesion coatings with aqueous lubricating layer, cryopreservation of cells by inhibiting ice recrystallization, and two-dimensional (2D) and three-dimensional (3D) porous materials with tunable pore sizes through recrystallized ice crystal templates. This Account sheds new light on the molecular mechanism of ice formation and will inspire the design of unprecedented functional materials based on controlled ice formation.

Control of ice nucleation: freezing and antifreeze strategies.

Water freezing remains a perennial topic of great relevance to many important aspects of our lives; from the climate to human society and from economics to medicine, frozen water profoundly influences our living environment and life activities. There have been numerous publications on water freezing; however, confusion regarding the process of freezing remains. In this review, we mainly focused on the nucleation aspects of water freezing; in particular, we focused on the effect of the surface morphology and nanostructure of foreign bodies. This review covers the recent progress in ice nucleation and anti-freezing strategies within the framework of nucleation principles. In this regard, we first summarize the crystal nucleation theories. Due to high interfacial energy, ice crystallization is primarily controlled by heterogeneous nucleation events, because the homogeneous nucleation barrier of ice is extremely high. In addition to the interfacial energy, the interfacial morphology or nanostructure of foreign bodies plays a diverse role under different supercooling regimes due to the Gibbs-Thomson effect. This effect gives rise to the inverse homogeneous-like nucleation phenomenon, in which foreign bodies have little influence on the nucleation barrier. This ensures the accurate measurement of the nucleation barrier, critical size, and water-ice interfacial energy, in agreement with the latest studies based on a microemulsions approach, metadynamics, the mW model, etc. As a consequence, anti-freezing strategies can be implemented by reducing the nucleation rate through restriction of the contact area of the water/substrate interface, by increasing the heterogeneous nucleation barrier through modification of the interfacial properties of foreign particles, including the interfacial structure and the interaction between the water and foreign particles and by kink kinetics. Within this context, the anti-freezing mechanism of superhydrophobic substrates was reviewed. Therefore, it follows that by significantly reducing the contact area between the water and substrate, superhydrophobic materials can effectively reduce the heterogeneous nucleation rate. We hope that this review will provide a unified picture and guidance for future work on water freezing.

Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb). Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies. mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried. The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.

Effect of Controlled Ice Nucleation on Stability of Lactate Dehydrogenase During Freeze-Drying

Several controlled ice nucleation techniques have been developed to increase the efficiency of the freeze-drying process as well as to improve the quality of pharmaceutical products. Owing to the reduction in ice surface area, these techniques have the potential to reduce the degradation of proteins labile during freezing. The objective of this study was to evaluate the effect of ice nucleation temperature on the in-process stability of lactate dehydrogenase (LDH). LDH in potassium phosphate buffer was nucleated at −4°C, −8°C, and −12°C using ControLyo™ or allowed to nucleate spontaneously. Both the enzymatic activity and tetramer recovery after freeze-thawing linearly correlated with product ice nucleation temperature (n = 24). Controlled nucleation also significantly improved batch homogeneity as reflected by reduced inter-vial variation in activity and tetramer recovery. With the correlation established in the laboratory, the degradation of protein in manufacturing arising from ice nucleation temperature differences can be quantitatively predicted. The results show that controlled nucleation reduced the degradation of LDH during the freezing process, but this does not necessarily translate to vastly superior stability during the entire freeze-drying process. The capability of improving batch homogeneity provides potential advantages in scaling-up from lab to manufacturing scale.

Optimization of freezing parameters for freeze-drying mango (Mangifera indica L.) slices

ABSTRACTToday, there is an increasing trend toward consuming healthful food products, and dried fruits are one option for obtaining desirable nutrition in dehydrated products. Proper drying method selection is important for minimizing quality losses. Lyophilization (freeze-drying), a technique that removes water by sublimation at low temperature, can produce excellent dried products. Fast vacuum induction during freezing has been proposed as a way to control ice nucleation and to obtain enhanced lyophilized pharmaceutical products. However, this technique has not yet been applied to food freeze-drying. The aim of this work was to evaluate the effects of the freezing rate (0.4, 0.25, and 0.1°C/min), the minimum chamber pressure during vacuum-induced surface freezing (900, 700, and 500 mTorr) and the sample temperature at which the induced vacuum was applied (0, −2, and −4°C) on the total process time, final moisture content, rehydration capacity, total color difference, and total polyphenol content during the lyophilization of mango (Mangifera indica L.) slices. A Box–Behnken design with three factors at three levels was used to design the experiments, to generate the polynomial equations relating the dependent and independent variables, and to determine the optimal operational conditions. The results highlight the reduction of total process time (30%) at high freezing rates and the influence of the tested operating conditions on freeze-drying optimization. It was found that the optimal conditions that satisfy commercial quality goals were pressure between 500 and 650 mTorr, temperature between −2 and 0°C, and freezing rate values close to 0.4°C/min.

2017_Review on the control of ice nucleation by ultrasound waves, electric and magnetic fields

2017_Review on the control of ice nucleation by ultrasound waves, electric and magnetic fields