Polar environments pose extreme challenges for life due to low temperatures, limited water, high radiation, and frozen landscapes. Despite these harsh conditions, numerous macro and microorganisms have developed adaptive strategies to reduce the detrimental effects of extreme cold. A primary survival tactic involves avoiding or tolerating intra and extracellular freezing. Many organisms achieve this by maintaining a supercooled state by producing small organic compounds like sugars, glycerol, and amino acids, or through increasing solute concentration. Another approach is the synthesis of ice-binding proteins, specifically antifreeze proteins (AFPs), which hinder ice crystal growth below the melting point. This adaptation is crucial for preventing intracellular ice formation, which could be lethal, and ensuring the presence of liquid water around cells. AFPs have independently evolved in different species, exhibiting distinct thermal hysteresis and ice structuring properties. Beyond their ecological role, AFPs have garnered significant attention in biotechnology for potential applications in the food, agriculture, and pharmaceutical industries. This review aims to offer a thorough insight into the activity and impacts of AFPs on water, examining their significance in cold-adapted organisms, and exploring the diversity of microbial AFPs. Using a meta-analysis from cultivation-based and cultivation-independent data, we evaluate the correlation between AFP-producing microorganisms and cold environments. We also explore small and large-scale biotechnological applications of AFPs, providing a perspective for future research.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype.
RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain\'s adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage.
CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain\'s safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

Advancements in extracellular vesicle (EV) studies necessitate the development of optimized storage conditions to ensure preservation of physical and biochemical characteristics. In this study, the most common buffer for EV storage (phosphate-buffered saline/PBS) was compared to a cryoprotective 5% sucrose solution. The size distribution and concentration of EVs from two different sources changed to a greater extent after -80 °C storage in PBS compared to the sucrose solution. Additionally, molecular surface protrusions and transmembrane proteins were more prevalent in EVs stored in the sucrose solution compared to those stored in PBS. This study demonstrates, for the first time, that distinct ring-like molecular complexes and cristae-like folded membranous structures are visible upon EV degradation. Taken together, the size, concentration, molecular surface extensions, and transmembrane proteins of EVs varied substantially based on the buffer used for -80 °C storage, suggesting that biocompatible cryoprotectants, such as sucrose, should be considered for EV studies.

Biological cryopreservation often involves using a cryoprotective agent (CPA) to mitigate lethal physical stressors cells endure during freezing and thawing, but effective CPA concentrations are cytotoxic. Hence, natural polysaccharides have been studied as biocompatible alternatives. Here, a subset of 26 natural polysaccharides of various chemical composition was probed for their potential in enhancing the metabolic post-thaw viability (PTV) of cryopreserved Vero cells. The best performing cryoprotective polysaccharides contained significant fucose amounts, resulting in average PTV 2.8-fold (up to 3.1-fold) compared to 0.8-fold and 2.2-fold for all non-cryoprotective and cryoprotective polysaccharides, respectively, outperforming the optimized commercial CryoStor™ CS5 formulation (2.6-fold). Stoichiometrically, a balance between fucose (18-35.7 mol%), uronic acids (UA) (13.5-26 mol%) and high molecular weight (MW > 1 MDa) generated optimal PTV. Principal component analysis (PCA) revealed that fucose enhances cell survival by a charge-independent, MW-scaling mechanism (PC1), drastically different from the charge-dominated ice growth disruption of UA (PC2). Its neutral nature and unique properties distinguishable from other neutral monomers suggest fucose may play a passive role in conformational adaptability of polysaccharide to ice growth inhibition, or an active role in cell membrane stabilization through binding. Ultimately, fucose-rich anionic polysaccharides may indulge in polymer-ice and polymer-cell interactions that actively disrupt ice and minimize lethal volumetric fluctuations due to a balanced hydrophobic-hydrophilic character. Our research showed the critical role neutral fucose plays in enhancing cellular cryopreservation outcomes, disputing previous assumptions of polyanionicity being the sole governing predictor of cryoprotection.

The aim of the present study was to evaluate semen cryopreservation with ACP-Lact® diluent, which consists of coconut water powder (ACP) added to goat milk powder. After thawing, the samples were evaluated for sperm kinetics, membrane evaluation and in vivo insemination. For cryopreservation, a pool was made with the ejaculate of six goats, diluted in four equal aliquots for the respective treatments: T1 (ACP-Lact®); T2 (ACP-Lact® 50%); T3 (ACP + 2.5% egg yolk) and T4 (Tris + 2.5% egg yolk). After dilution of the treatments, the samples were placed in 0.5 ml straws and chilled at a rate of -1.07°C/min. After reaching 4°C and stabilizing for one hour, the straws were placed in nitrogen vapour at -60°C for 15 minutes and then immersed in liquid nitrogen (-196ºC). The straws were thawed in a 37°C water bath and kinetic assessments were performed immediately using a computerized semen analysis program (CSA), viability (EN), membrane functionality (HOST), mitochondrial activity (DAB) and DNA integrity assessment of spermatozoa. For the in vivo experiment, ten goats were inseminated, divided into two groups of five goats each, G1 inseminated with ACP-Lact® and G2 with ACP, by fixed-time artificial insemination (FTAI). Regarding the kinetic parameters, the ACP-Lact® treatment showed higher progressive motility (PM) and sperm velocity than the other treatments (36.77%). In the VSL parameter the ACP-Lact diluent was superior to ACP and Tris. In viability the treatment with ACP-Lact® was superior to the treatment with Tris, 95% and 83% respectively. In FTAI two goats were born out of the 5 goats inseminated with ACP-Lact®. It was concluded that the use of ACP-Lact® for cryopreservation of caprine semen is efficient in maintaining seminal parameters during thawing in vitro and in vivo and proved to be a good alternative extender for the caprine species.

Carbohydrates are a class of macromolecules that has significant potential across several domains, including the organisation of genetic material, provision of structural support, and facilitation of defence mechanisms against invasion. Their molecular diversity enables a vast array of essential functions, such as energy storage, immunological signalling, and the modification of food texture and consistency. Due to their rheological characteristics, solubility, sweetness, hygroscopicity, ability to prevent crystallization, flavour encapsulation, and coating capabilities, carbohydrates are useful in food products. Carbohydrates hold potential for the future of therapeutic development due to their important role in sustained drug release, drug targeting, immune antigens, and adjuvants. Bio-based packaging provides an emerging phase of materials that offer biodegradability and biocompatibility, serving as a substitute for traditional non-biodegradable polymers used as coatings on paper. Blending polyhydroxyalkanoates (PHA) with carbohydrate biopolymers, such as starch, cellulose, polylactic acid, etc., reduces the undesirable qualities of PHA, such as crystallinity and brittleness, and enhances the PHA\'s properties in addition to minimizing manufacturing costs. Carbohydrate-based biopolymeric nanoparticles are a viable and cost-effective way to boost agricultural yields, which is crucial for the increasing global population. The use of biopolymeric nanoparticles derived from carbohydrates is a potential and economically viable approach to enhance the quality and quantity of agricultural harvests, which is of utmost importance given the developing global population. The carbohydrate biopolymers may play in plant protection against pathogenic fungi by inhibiting spore germination and mycelial growth, may act as effective elicitors inducing the plant immune system to cope with pathogens. Furthermore, they can be utilised as carriers in controlled-release formulations of agrochemicals or other active ingredients, offering an alternative approach to conventional fungicides. It is expected that this review provides an extensive summary of the application of carbohydrates in the realms of food, pharmaceuticals, and environment.

For the safety and efficacy of frozen cell therapy products, determination of cellular viability is key. However, results of cell viability measurements do not only depend on the cell line or on the inflicted stress, but also on the assay used, making inter-experimental comparisons difficult. The aim of this study was thus to assess commonly used viability assays in clinically relevant human mesenchymal/stromal stem cells and human A549 lung carcinoma cells. Post freeze-thaw stress viability and proliferation were evaluated under different conditions using trypan blue, acridine orange/DAPI stain, alamarBlue, ATP, and neutral red assays. Significant differences in cell viability between metabolic assays were observed, likely due to their distinct intrinsic detection mechanisms. Membrane-integrity based assays generally overestimated cell viabilities in this study. Furthermore, noticeable differences in inter-assay sensitivities were observed. These differences highlight that cell viability methods should be meticulously selected and their associated results carefully interpreted in a relevant context to ensure reliable conclusions. Indeed, although cell membrane integrity based assays are a popular choice to determine cellular quality attributes after freezing and thawing, we demonstrate that metabolic assays may be more suitable in this context.

Polysaccharides are becoming potential candidates for developing food-grade cryoprotectants due to their extensive accessibility and health-promoting effects. However, unremarkable ice recrystallization inhibition (IRI) activity and high viscosity limit their practical applications in some systems. Our previous study found a galactoxyloglucan polysaccharide from tamarind seed (TSP) showing moderate IRI activity. Herein, the enhancement of the IRI performance of TSP via enzymatic depolymerization and degalactosylation-induced self-assembly was reported. TSP was depolymerized and subsequently removed ∼40 % Gal, which induced the formation of supramolecular rod-like fiber self-assembles and exhibited a severalfold enhancement of IRI. Ice shaping assay did not show obvious faceting of ice crystals, indicating that both depolymerized and self-assembled TSP showed very weak binding to ice. Molecular dynamics simulation confirmed the absence of molecular complementarity with ice. Further, it highlighted that degalactosylation did not cause significant changes in local hydration properties of TSP from the view of a single oligomer. The inconsistency between molecular simulation and macroscopic IRI effect proposed that the formation of unique supramolecular self-assemblies may be a key requirement for enhancing IRI activity. The findings of this study provided a new opportunity to enhance the applied potential of natural polysaccharides in food cryoprotection.

Polymeric micelles are promising carriers for the delivery of poorly water-soluble drugs, providing enhanced drug solubility, blood circulation times, and bioavailability. Nevertheless, the storage and long-term stability of micelles in solution present challenges requiring the lyophilization and storage of formulations in the solid state, with reconstitution immediately prior to application. Therefore, it is important to understand the effects of lyophilization/reconstitution on micelles, particularly their drug-loaded counterparts. Herein, we investigated the use of β-cyclodextrin (β-CD) as a cryoprotectant for the lyophilization/reconstitution of a library of poly(ethylene glycol-b-ε-caprolactone) (PEG-b-PCL) copolymer micelles and their drug-loaded counterparts, as well as the effect of the physiochemical properties of different drugs (phloretin and gossypol). The critical aggregation concentration (CAC) of the copolymers decreased with increasing weight fraction of the PCL block (fPCL), plateauing at ~1 mg/L when the fPCL was >0.45. The blank (empty) and drug-loaded micelles were lyophilized/reconstituted in the absence and presence of β-CD (9% w/w) and analyzed via dynamic light scattering (DLS) and synchrotron small-angle X-ray scattering (SAXS) to assess for changes in aggregate size (hydrodynamic diameter, Dh) and morphology, respectively. Regardless of the PEG-b-PCL copolymer or the use of β-CD, the blank micelles displayed poor redispersibility (<10% relative to the initial concentration), while the fraction that redispersed displayed similar Dh to the as-prepared micelles, increasing in Dh as the fPCL of the PEG-b-PCL copolymer increased. While most blank micelles displayed discrete morphologies, the addition of β-CD or lyophilization/reconstitution generally resulted in the formation of poorly defined aggregates. Similar results were also obtained for drug-loaded micelles, with the exception of several that retained their primary morphology following lyophilization/reconstitution, although no obvious trends were noted between the microstructure of the copolymers or the physicochemical properties of the drugs and their successful redispersion.

Reactive oxygen species (ROS) and oxidative stress (OS), the imbalance between the production of free radicals and the cellular antioxidant defenses, are discussed in relation to their role in bovine sperm physiology. Oxidative stress has been associated to male infertility and low fertility rates in Assisted Reproductive Techniques (ART). Antioxidant supplementation is an interesting approach to overcome OS-related infertility and assisted reproduction drawbacks. Several studies have been conducted to identify the potential sources of ROS in a typical ART setting and the impact of antioxidant supplementation on semen quality and pregnancy outcome. Procedures such as freezing and thawing, centrifugation and incubation are thought to produce significant amounts of ROS with a negative impact on sperm quality parameters and reproductive competence. Given the important role of ROS in sperm function, the addition of antioxidants in sperm media to prevent OS and to improve the reproductive outcome requires attention. Currently, there is limited evidence to support the ameliorative effect of antioxidant supplementation on fertilization and embryo development in farm animals. This review summarizes the different types and concentrations of antioxidants used in sperm preparation media of bovine species and their effectiveness in neutralizing excessive ROS production while preserving physiological sperm function.