Microwave-assisted Dehydration Research Articles

Microwave-assisted dehydration, long-term storage at non-freezing temperatures, and rehydration of cat germinal vesicles.

Germinal vesicles (GVs) are alternative targets for female fertility preservation due to their availability and high resilience against non-physiological conditions. Preserved GVs can then be transferred to fresh cytoplasts to reconstitute viable oocytes. Here, we describe a GV preservation method that employs non-ionizing microwave radiations imparting energy to water molecules, which results in rapid and homogeneous drying of the sample. Trehalose is added as a xero-protectant before the radiations, enabling isothermal vitrification of the disaccharide sugar during drying. While the technique is still considered experimental, studies have shown that DNA and structural integrity can be effectively maintained in dried/rehydrated GVs. Importantly, the dry-preservation approach allows supra-zero temperature storage of the samples, offering a cost-effective and energy-saving alternative to traditional methods relying on ultra-low freezing temperatures. The protocol outlines a comprehensive procedure involving GV oocyte collection, trehalose loading, microwave drying, storage, and rehydration. The simplicity of the protocol facilitates ease of manipulation, making it an accessible method for researchers. While initially developed for domestic cats, the protocol can be adapted for other species with necessary modifications, considering potential species-specific responses to dehydration stress.

The Effect of Choline Salt Addition to Trehalose Solution for Long-Term Storage of Dried and Viable Nuclei from Fully Grown Oocytes.

Although drying techniques are exciting alternatives to cryopreservation, it remains challenging to maintain tightly controlled temperatures and humidity levels during storage of dried products. The objective of this study was to determine if the addition of choline acetate to trehalose solution could enable a wider range of storage conditions for preservation of nuclei from fully grown oocytes, by allowing temporary humidity excursions (>44% relative humidity) that may lead to crystallization of trehalose and loss of DNA integrity. Using domestic cat germinal vesicle oocytes as a model, we characterized the recovery as well as the integrity of samples after microwave-assisted dehydration. Exposure to choline acetate alone did not impair the germinal vesicle's DNA integrity and only had a negative impact on the chromatin configuration. Choline acetate addition enabled us to reach lower moisture contents after 25 min of microwave-assisted drying. Sample recovery after rehydration was also better in the presence of choline acetate. The integrity of the germinal vesicle's DNA was not affected, while the chromatin configuration was impaired by the presence of choline acetate during dehydration. Importantly, choline acetate addition helped to maintain an amorphous state (absence of detrimental crystallization) during excursion from ideal humidity conditions.

O-173 Cellular and developmental damage from oocyte cryopreservation

Abstract Compared to somatic or sperm cells, mammalian oocytes are much more sensitive to cryopreservation mainly because of the large volume, cytoskeleton, and presence of the zona pellucida. Connections with the surrounding cumulus cells also are challenging to preserve in immature oocytes. Besides the genetic and epigenetic information enclosed in the nucleus, oocytes contain organelles and cytoplasmic factors that are necessary for the early embryo development. Therefore, exposure to cryoprotectant and freezing temperatures can easily damage the oocyte’s complex cellular structure and developmental competence, which leads to poor success in fertilization, lower embryo quality, and reduced pregnancy rates. Using the domestic cat as a model, our laboratory has studied damages in cumulus-oocyte complexes (immature oocytes) occurring at each step of the cryopreservation process. Specifically, this included the impact of cryoprotectants on microtubules, the effect of osmotic changes on the overall structures, as well as sensitivity of the nuclear chromatin and epigenetic patterns to freezing temperatures. Based on those findings, it has been possible to design mitigating solutions like customized ultra-rapid freezing or vitrification protocols, Laser-warming methods, or post-warming reanimations. New horizons have also been explored to move away from issues and limitations related to cryopreservation. Approaches like storage of oocytes for the long-term at non-freezing temperatures are currently being developed based on encouraging data generated in preliminary studies. For instance, incorporation of trehalose into the oocyte followed by microwave-assisted dehydration can allow to remove enough water while creating a protective trehalose glass compatible with survival and storage at non-freezing temperatures. Interestingly, transcriptomic and proteomic studies have already shown that stresses and damages induced by dehydration versus cryopreservation are different and may be easier to overcome.

Trehalose delivered by cold-responsive nanoparticles improves tolerance of cumulus-oocyte complexes to microwave drying.

Trehalose is a non-permeable protectant that is the key to preserve live cells in a dry state for potential storage at ambient temperatures. After intracellular trehalose delivery via cold-responsive nanoparticles (CRNPs), the objective was to characterize the tolerance of cat cumulus-oocyte complexes (COCs) to different levels of microwave-assisted dehydration. Trehalose was first encapsulated in CRNPs. After exposure to trehalose-laden CRNPs, different water amounts were removed from cat COCs by microwave drying. After each dehydration level, meiotic and developmental competences were evaluated via in vitro maturation, fertilization, and embryo culture. In addition, expressions of critical genes were assessed by quantitative RT-PCR. CRNPs effectively transported trehalose into COCs within 4h of co-incubation at 38.5°C followed by a cold-triggered release at 4°C for 15min. Intracellular presence of trehalose enabled the maintenance of developmental competence (formation of blastocysts) as well as normal gene expression levels of HSP70 and DNMT1at dehydration levels reaching up to 63% of water loss. Intracellular trehalose delivery through CRNPs improves dehydration tolerance of COCs, which opens new options for oocyte storage and fertility preservation at ambient temperatures.

Feline microRNAome in ovary and testis: Exploration of in-silico miRNA-mRNA networks involved in gonadal function and cellular stress response.

The aim of the study was to perform the first in-depth analysis of miRNAs in ovarian and testicular tissues of the domestic cat, a critical biomedical model. Specifically, potential miRNA involvement was explored in gonadal function, testis development, and cellular stress response to preservation protocols. We performed miRNA-sequencing on 20 ovarian and 20 testicular samples from 15 cats, including different ages and tissue treatments. Using fresh tissues (n = 15), we confirmed gonadal expression of 183 miRNA precursors and discovered additional 52 novel feline candidate precursors. We integrated the mRNA data from our previous study on the same age and treatment groups to create in-silico miRNA-mRNA networks and their functional enrichment, which allows comprehensive exploration into possible miRNA functions in cat gonads. Clusters of miRNAs united by shared differentially expressed mRNA targets are potentially involved in testicular development and spermatogenesis. MicroRNAs could play a significant role in ovarian tissue response to stress from microwave-assisted dehydration, with smaller roles in cellular response to vitrification in both ovary and testis. This new list of miRNAs with potential function in cat gonads is a major step towards understanding the gonadal biology, as well as optimizing fertility preservation protocols.

Advanced osmotic dehydration techniques combined with emerging drying methods for sustainable food production: Impact on bioactive components, texture, color, and sensory properties of food.

The food industries are looking for potential preservation methods for fruits and vegetables. The combination of osmosis and drying has proved the efficient method to improve the food quality. Osmotic dehydration is a mass transfer process in which water molecules from the food move to an osmo-active solution and the solutes from the solution migrate into the food. Advanced osmotic dehydration techniques such as electric field pulse treatment, ultrasonic and microwave-assisted dehydration, pulsed vacuum, and osmodehydrofreezing can improve the nutritional quality (bioactive) and sensory properties (color, texture, aroma, flavor) of fresh and cut-fruits without changing their reliability. Emerging osmotic dehydration technologies can preserve the structure of fruit tissue by forming microscopic channels and increasing effective water diffusivity. However, it is important to analyze the effect of advanced osmotic dehydration techniques on the quality of food products to understand the industrial scalability of these techniques. The present paper discusses the impact of recent osmotic dehydration techniques on bioactive, antioxidant capacity, color, and sensory profile of food.

Microwave-assisted preparation of polyphosphoric acid in a continuous-flow reactor

Abstract Polyphosphoric acid (PPA) is widely used in inorganic salt production, petrochemical industry, electronic material preparation and other manufacturing industries. Conventional preparation methods of PPA has disadvantages of pollution, high energy consumption and long production time. To address this problem, microwave continuous-flow preparation may be a desirable way due to its advantages of environmentally-friendly, rapidity and high efficiency. Therefore, to explore the process of preparing PPA by microwave continuous-flow method, a continuous-flow microwave reactor was designed for the dehydration process of orthophosphoric acid to prepare PPA in this paper. The microwave-assisted dehydration process was studied in comparison with the conventional dehydration process and the “closed” microwave-assisted dehydration process in terms of energy efficiency, process times and treatment capacity. The effect of input microwave power, reduced pressure and inlet flow velocity of orthophosphoric acid on the performance of the dehydration process was studied. The results showed that the influence of the microwave power on the temperature rise process during dehydration is greater than that of the reduced pressure. Moreover, the inlet flow rate has a great impact on the treatment capacity and product quality of the dehydration process. Bedsides, the comparison with the other two methods showed that microwave heating can effectively shorten the dehydration time, and the continuous-flow treatment can effectively improve the treatment capacity of microwave heating. The perspectives of the process scale-up by continuous-flow microwave heating method is also discussed.

Initial response of ovarian tissue transcriptome to vitrification or microwave-assisted dehydration in the domestic cat model

BackgroundLong term preservation of living ovarian tissues is a critical approach in human reproductive medicine as well as in the conservation of rare animal genotypes. Compared to single cell preservation, optimization of protocols for tissues is highly complex because of the diversity of cells responding differently to non-physiological conditions. Using the prepubertal domestic cat as a model, the objective was to study immediate effects of vitrification or microwave-assisted dehydration on the global transcriptome dynamics in the ovarian cortex. RNA sequencing was performed on ovarian tissues (n = 6 individuals) from different conditions: fresh tissue after dissection (F), vitrified/warmed tissue (V), tissue dehydrated for 5 min (D5) or 10 min (D10) followed by rehydration. Differential gene expression analysis was performed for comparison pairs V vs. F, D10 vs. F, D5 vs. F and D10 vs. D5, and networks were built based on results of functional enrichment and in silico protein-protein interactions.ResultsThe impact of the vitrification protocol was already measurable within 20 min after warming and involved upregulation of the expression of seven mitochondrial DNA genes related to mitochondrial respiration. The analysis of D10 vs. F revealed, 30 min after rehydration, major downregulation of gene expression with enrichment of in silico interacting genes in Ras, Rap1, PI3K-Akt and MAPK signaling pathways. However, comparison of D5 vs. F showed negligible effects of the shorter dehydration protocol with two genes enriched in Ras signaling. Comparison of D10 vs. D5 showed downregulation of only seven genes. Vitrification and dehydration protocols mainly changed the expression of different genes and functional terms, but some of the differentially expressed genes formed a major in silico protein-protein interaction cluster enriched for mitochondrial respiration and Ras/MAPK signaling pathways.ConclusionsOur results showed, for the first time, different effects of vitrification and microwave-assisted dehydration protocols on the global transcriptome of the ovarian cortex (using the domestic cat as a biomedical model). Acquired data and networks built on the basis of differentially expressed genes (1) can help to better understand stress responses to non-physiological stresses and (2) can be used as directions for future preservation protocol optimizations.

Influence of microwave-assisted dehydration on morphological integrity and viability of cat ovarian tissues: First steps toward long-term preservation of complex biomaterials at supra-zero temperatures.

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues.

Modelling and Kinetic Study of Novel and Sustainable Microwave-Assisted Dehydration of Sugarcane Juice

Sugarcane juice is a perishable food with a good nutritional profile. Thus, there is a need to increase its shelf life by reducing water content which facilitates storage and transportation. In this study, process conditions were optimized to concentrate the sugarcane juice at various microwave powers (30, 50, 80, 100 W). A central composite design was applied to optimize the process conditions (power and time). The overall evaporation time depends on microwave powers; increase in power reduced the processing time. The results showed that at 100 W sugarcane juice was concentrated to 75° brix for 15 min which reduced the energy consumption to 1.3 times compared to other powers. Moreover, microwave processing better retained the sensory properties of concentrate and preserved its antioxidant activity. Thus, 100 W was most energy efficient in concentrating sugarcane juice. In general, microwave processing reduced the processing time and cost making it a sustainable approach to concentrate juices.

Functionalized zeolite-solvent catalytic systems for microwave-assisted dehydration of fructose to 5-hydroxymethylfurfural

Abstract This study investigates the structure-performance relationships in different zeolite-solvent systems that are suitable for microwave-assisted dehydration of food waste-derived fructose to 5-hydroxymethylfurfural (HMF). Different types of zeolites (MFI, BEA, and Y) were examined as acid catalysts. Water was first tested as the greenest solvent (baseline), followed by dimethyl sulfoxide (DMSO), acetone, γ-valerolactone (GVL), and propylene carbonate (PC)/water (1:1 v/v) binary solvent systems. The results showed that the HMF yield was independent of particle size of MFI zeolite in water. The secondary porosities improved the HMF yield, while byproducts formation (via rehydration or polymerization) was also increased due to the enlarged channels in zeolites. All tested zeolites showed higher fructose conversion, HMF yield, and HMF selectivity in organic-water solvent systems than in water. The synergistic effects of the substrates, catalysts, and solvent-product interactions in the hydrophobic Y zeolite/DMSO system yielded the highest fructose conversion (72.4%) and HMF yield (49.2%). This study advances our understanding of green solvents and functionalized zeolites for microwave-assisted biomass conversion in sustainable biorefinery.

Mesoporous titanium dioxide nanocatalyst: a recyclable approach for one‐pot synthesis of 5‐hydroxymethylfurfural

The present study deals with the production of 5-hydroxymethyl furfural (HMF) from fructose by chemo-conversion method using chemical catalyst, conventionally achieved by microwave-assisted dehydration process. Five different chemical catalysts, namely oxalic acid, phosphotungstic acid and mesoporous titanium dioxide nanoparticles (TNPs) were compared at constant conditions of which TNPs yielded a maxima of 33.95%. The optimum temperature and catalyst loading were found to be 200°C and 20%, respectively, at a 5% optimum substrate concentration during 15 min optimum reaction time to yield 61.53% HMF. The efficiency of synthesised TNPs was investigated further through reusability studies. TNPs were properly recycled and the catalytic activity recovery was good even after a 14 batch reactions. The specific surface area of the TNP obtained is about 105.46 m2/g and its pore-volume is about 0.42 cm3/g according to single point adsorption. A large accessible surface area combined with a minimal pore size (15.92 nm) obtained with mesoporous TNPs is desirable for better catalyst loading, high-yield HMF, retention and reduced diffusion constraints.

Microwave-assisted dehydration of fructose and inulin to HMF catalyzed by niobium and zirconium phosphate catalysts

5-Hydroxymethyl-2-furaldehyde (HMF) is a key bio-based platform for the production of renewable monomers and bio-fuels. However, most of its syntheses are carried out under not sustainable conditions. In this work, the production of HMF from fructose and inulin was investigated following the Green Chemistry principles, adopting aqueous medium, appreciable substrate concentration (10wt%), low loading of heterogeneous acid catalyst (niobium or zirconium phosphate) and microwave heating. Both the catalysts resulted very active and promising, in particular zirconium phosphate and the performances were related to their different acid characteristics. The optimization of HMF synthesis with zirconium phosphate was also supported by a statistical modelling, which shows that the highest yield to HMF (about 40mol%) is ascertained at high temperature (190°C) and short reaction time (8min). The catalysts resulted recyclable maintaining their starting activity almost unchanged.

Conversion of xylose, xylan and rice husk into furfural via betaine and formic acid mixture as novel homogeneous catalyst in biphasic system by microwave-assisted dehydration

Dehydration of d-xylose and direct transformation of xylan into furfural were achieved by means of betaine-formic acid (HCOOH) catalytic system. All reactions were microwave-assisted and carried out in a CPME-water biphasic system. At 170°C, in a pH range between 1.9 and 2.3, highest yields of 80% and 76% were obtained respectively for the pentose and the polysaccharide. Time dependence of the dehydration and influence of the temperature on the reaction kinetics were studied. Besides, at 190°C, using the optimized condition of the reaction, rice husk was also employed as a source of furfural with a single stage reaction.

Microwave-assisted dehydration of D-xylose into furfural by diluted inexpensive inorganic salts solution in a biphasic system

Production of furfural from biomass using less corrosive acids and minimizing the formation of undesired by-products is great challenge. Dehydration of D-xylose to furfural was carried out under microwave irradiation in biphasic system consisting of CPME and aqueous inorganic salts solution. Best conditions of dehydration of D-xylose using FeCl3 were investigated by varying temperature, reaction time, pentose loading and influence of NaCl. Highest furfural yield of 74% was achieved at 170°C for 20min in presence of 10mol% of iron chloride and 100mol% of sodium chloride in water-CPME, 1:3, v/v. Addition of NaCl was found to increase the catalytic activity of FeCl3 and allowed to reduce the amounts of FeCl3 used from 20mol% to 10mol%. This approach was tested with success starting from L-arabinose and xylan, a pentose-rich polysaccharide.

Synthesis of Phenylacetaldehyde from 1-Phenylethan-1,2-diol by Microwave-Assisted Dehydration in Water

A highly efficient, simple and versatile acid catalyst is proposed for the selective dehydration of 1-phenylethan-1,2-diol to the phenylacetaldehyde as an industrial platform molecule. For the first time, common Lewis acids AlCl3 and FeCl3 and mineral acids HCl and H2SO4 had produced the acetaldehyde derivative under microwave irradiation in sub-critical water in good yield starting from the corresponding diol.

Resilience of oocyte germinal vesicles to microwave-assisted drying in the domestic cat model.

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased (p<0.05) from 1.7 to 0.1 gH2O/gDW over the first 30 min, but did not decrease further (p>0.05) after 40 min. Chromatin configuration was unaffected (p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered (p>0.05) from 0 to 30 min of treatment (range, 6.1%-12%), but increased (p<0.05) to 32.5% after 40 min. Next, the influence of storage at two different supra-zero temperatures after 30 min of drying was investigated. Oocyte-loaded, microwave-treated filters were individually sealed in Dri-Shield moisture barrier bags and stored at 4°C or ambient temperature for 0 to 8 weeks. Moisture contents gradually decreased (p<0.05) from 0.12 to 0.10 gH2O/gDW after 8 weeks of storage at 4°C or ambient temperature. The percentage of GVs with DNA fragmentation more than doubled (p<0.05) from 0 (14.3%) to 2 days (30.0%-33.0%), but remained stable (p>0.05) thereafter (1 through 4 weeks, 25.0%-35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature.

086 Optimal preservation of DNA Integrity in cat germinal vesicles after microwave-assisted dehydration and supra-zero temperature storage

The germinal vesicle (GV) is an alternative target to the whole oocyte for exploring new genome preservation approaches at supra-zero temperatures. Using the domestic cat model, we have demonstrated that the GV can survive simple air-drying and then be hydrated to reconstitute a viable oocyte (Graves-Herring et al., 2013). However, this method can lead to variable DNA damage due to uncontrolled water loss during the dehydration and storage processes. The objective of the present study was to better understand the role of water content during these critical steps on GV DNA integrity. Intraovarian immature oocytes collected from adult cat ovaries were denuded and permeabilized before exposure to 1.5 M trehalose for 10 min. Oocytes were transferred onto conjugate-release filters and dehydrated with a CEM SAM 255 microwave system. In Experiment 1, samples (n = 337 total oocytes, 4–5 replicates) were dehydrated at 20% power for 0, 5, 10, 15, 20, 25, 30 or 40 min. Moisture contents were assessed immediately after microwaving using volumetric Karl Fischer titration. Oocytes were rehydrated to assess chromatin configuration (Hoechst staining) and DNA fragmentation (TUNEL assay). Moisture contents progressively decreased (P 0.05) after 40 min. Chromatin configuration and proportions of degenerated oocytes (range, 2.2–9.1%) were unaffected (P > 0.05) over time. The incidence of GVs with DNA fragmentation was unaltered (P > 0.05) from 0 to 30 min of treatment (range, 6.1–12%), but increased (P 0.05) thereafter (1 through 4 weeks, 25.0–35.0%). This study characterizes, for the first time, the metrics and kinetics of moisture removal, degeneration and DNA damage related to GV desiccation as an alternative approach for preserving the maternal genome at cool or ambient temperature. Findings demonstrate the feasibility for successful microwave dehydration of the mammalian GV to reach an equilibrium moisture content that is non-lethal. Most DNA disruption occurs within the first 2 days of storage at supra-zero temperatures and more than two-thirds of GVs retain intact DNA regardless of further moisture loss during storage. Source of funding: National Institutes of Health. Conflict of interest: None declared. comizzolip@si.edu

Beneficial energy-efficiencies in the microwave-assisted vacuum preparation of polyphosphoric acid

A vacuum microwave-assisted dehydration process of orthophosphoric acid for preparation of polyphosphoric acid was studied in comparison with the conventional dehydration in terms of energy efficiency and process rates. The influence of the applied microwave-power input on the process duration and the efficiency of microwave process were examined. The optimal microwave power input provided twofold shortening of the process time compared to conventional heating. At the same time the energy efficiencies remained comparable for both heating modes which was caused by nonoptimal filling ratio of the microwave cavity. The perspectives of the process scale-up in terms of increasing energy efficiency were also discussed.

Efficient microwave-assisted synthesis of 5-hydroxymethylfurfural from concentrated aqueous fructose

Studies on the HCl-catalysed microwave-assisted dehydration of highly concentrated aqueous fructose (27wt%) to 5-hydroxymethylfurfural (HMF) revealed a significant increase in the fructose conversion rate over the conventional heated systems. Water, being the most benign solvent and therefore ideal for green and sustainable chemistry, normally is a poor solvent for the dehydration process resulting in low HMF selectivities and yields. However, reaction at 200°C with microwave irradiation with a short reaction time of only 1s resulted in good HMF selectivity of 63% and fructose conversion of 52%, while prolonged irradiation for 60s (or more) resulted in nearly full fructose conversion (95%) but lower HMF yield (53%). Decreasing the fructose concentration significantly improved the HMF selectivity, but possibly made the production route less attractive from an industrial point of view due to the resultant low throughput.