Ice Crystals Research Articles

Effect of liquid nitrogen spray freezing on the ice crystal size and quality of large yellow croaker

Large yellow croakers (Larimichthys crocea) were frozen by liquid nitrogen spray freezing (LNSF), liquid immersion freezing (LIF), and air freezing (AF), and the freezing rate, ice crystal size and product qualities were compared. LNSF was found to be a promising freezing technology because of the significantly (P < 0.05) shortest freezing time, smallest ice crystal, and best overall product quality. Then, the effect of processing parameters during LNSF on freezing characteristics, and microstructure and qualities of samples was studied. Freezing time was reduced by decreasing freezing temperature and increasing air velocity. A size uniformity index was proposed in this study to evaluate the distribution uniformity of ice crystals. The minimum cross-sectional area (605.83 μm2) with the best size uniformity was achieved at a freezing temperature of −90 °C, a final temperature of −30 °C, and an air velocity of 4 m/s, showing a reduction of 50.90% as compared to the maximum (1233.77 μm2). The qualities of samples frozen by LNSF with the aforementioned parameter values were well maintained and met the industrial standard. Therefore, the developed LNSF protocol was recommended for the industrial application for large yellow croaker.

Pressure Shift Freezing: An Alternate Method of Freezing for Meat and Marine Products and Their Quality Characteristics

The comprehensive demand for meat and seafood in the global market has raised a severe need for processing and preservation strategies to conserve their inherent quality. It is widely perceived that the present conventional meat production system engages freezing as the primary preservation technique, and reports have documented several challenges associated with existing freezing approaches, like the creation of ice crystals during phase transition, which causes deterioration of the tissue membrane, destruction of the cell wall, and textural damage to the foods. Thus, interest in alternative food freezing methodologies, such as pressure shift freezing (PSF), has emerged as a matter of immense approaches and commercial interest owing to its massive benefits. This review delivers the fundamentals of PSF, including the mechanisms of ice crystal formation and microbial inactivation. Further, this review briefly states the applications of PSF in meat processing and preservation by compiling recent works. This provides insight into emerging trends and research requirements. High‐pressure freezing creates rapid and homogenous ice crystal development all over the food sample. Moreover, the impact of pressure on the quality aspect of PSF‐treated food was discussed. Also, the changes in functional characteristics of PSF‐treated foods like colour, texture, and WHC have been highlighted. Finally, aspects that can be further notably analysed, the obstruction that confines the implementation of this innovation on a large scale, and opportunities have been presented.

Unraveling the molecular freezing behavior of water on a calcium silicate hydrate matrix.

The hydration process of cement-based materials primarily results in the formation of calcium silicate hydrate (CSH), which is crucial in deciding how long concrete will last. This study utilizes molecular dynamics simulation technology to explore the freezing behavior of pure water solutions within various calcium silicate hydrate (CSH) matrices. The investigated matrices encompass four different Ca/Si ratios. According to the simulation, as ice crystals develop close to the surface of CSH, the CSH matrix strengthens its hydrogen and ionic interactions with water molecules, which effectively prevents water molecules from crystallizing and nucleating. Consequently, these molecules compose an unfrozen water film structure that bridges between ice crystals and the CSH matrix. The research also reveals an intriguing relationship between silica chain behavior on the Ca/Si ratio and the CSH surface. Surface flaws arise as a result of the silica chains of CSH breaking into shorter segments as the Ca/Si ratio increases. These surface defects manifest as grooves on the matrix's surface, effectively capturing and retaining specific water molecules. The CSH matrix's hydrogen bonds with water molecules are weakened as a result of this process, facilitating their participation in the crystallization process, and leading to a thinner unfrozen water film thickness with an increased Ca/Si ratio. This study contributes to a greater knowledge of the performance and dependability of cement-based products by offering molecular-level insights into the freezing actions of liquids in gel pores.

Freezing-enhanced chlorination of organic pollutants for water treatment.

Freezing has been reported to accelerate chemical reactions and thus affect the fate of pollutants in the environment. However, little research has been conducted on the potential effects of freezing on the chlorination process. This study aimed to explore the freezing-enhanced chlorination process by comparing the oxidation of clofibric acid (CA) by chlorine in ice (at -20 °C) to the same reaction in water (at 25 °C). The degradation of CA, which was negligible in water, was significantly accelerated in ice. This acceleration can be attributed to the freeze concentration effect that occurs during freezing, which excludes solutes such as chlorine, CA and protons from the ice crystals, leading to their accumulated concentration in the liquid brine. The increased concentration of chlorine and protons in the liquid brine leads to higher rates of CA oxidation, supporting the freeze concentration effect as the underlying cause for the accelerated chlorination of CA in ice. Moreover, the chlorine/freezing system was also effective in the degradation of other organic pollutants. This highlights the environmental relevance and significance of freezing-enhanced chlorination in cold regions, particularly for the treatment of organic contaminants.

Ice crystal guided folding of graphene oxides in a confined space: a facile approach to 1D functional graphene structures.

The controlled conformational changes of planar graphene nanosheets are of great importance to the realization of their practical applications. Despite substantial effort in the area, the controlled folding of two-dimensional (2D) graphene sheets into one-dimensional (1D) structures still remains a significant challenge. Here, for the first time, we report an ice crystal guided folding strategy to fabricate 1D folded graphene nanobelts (FGBs), where the formation and growth of ice crystals in a confined space function to guide the folding of 2D graphene oxide (GO) nanosheets into 1D nanobelts (i.e. folded graphene oxide belts, FGOBs), which were subsequently converted to FGBs after annealing. Thin aqueous GO containing films were obtained by blowing air through a GO dispersion in the presence of a surfactant, polyoxypropylenediamine (D400), resulting in a foam containing uniform air bubbles. Subsequent shock cooling of the foam using liquid nitrogen resulted in the facile fabrication of FGOBs. This technique provides a general approach to encapsulate catalytic nanomaterials such as Fe3O4 nanorods, TiO2 and Co3O4 nanoparticles into the folded graphene structure for practical applications such as Li-ion batteries.

Histological outcome evaluation of selected brain preparation protocols for white fiber dissection.

White fiber dissection is essential for studying brain connections. However, preparation protocols have not been validated. We microstructurally analyzed Klingler's brain preparation method and freezing process and assessed changes under two protocols:freeze-only and freeze-thaw. The microstructure changes of these protocols were evaluated by measuring the ratio of the total gap area to the white matter area and determining the mean eccentricity value to assess the degree of anisotropy. Sixty hemispheres were allocated to ten different freezing protocols. In the freeze-only protocols, the total gap area ratio was significantly higher compared to that of specimens fixed with only formaldehyde, particularly after continuous freezing for 3-4 weeks;however, the difference in eccentricity was not significant. In the freeze-thaw protocols, both the area ratio and eccentricity were significantly higher compared to the freeze-only. The optimum degree of fiber separation in the freeze-thaw protocols reached its peak with four cycles of 1-week freezing periods interrupted by six hours of thawing. The Klingler method assists in the separation of the white fibers through the gaps formed by ice crystals, but an appropriate degree of anisotropy is reached when the freezing protocol is interrupted by at least four thawing cycles.

An isochoric optical platform for interrogation of aqueous glass formation processes.

Aqueous vitrification (glass formation) processes are integral to modern cryopreservation, but experimental methods by which to study them are limited, particularly at the mL volume scales relevant to many biomedical applications. Here, we introduce an inexpensive custom optical platform, the isochoric vitrification cryo-macroscope (or "isovitriscope"), to supplement standard techniques with new qualitative and quantitative data streams. The platform consists of an LED light source, a isochoric (constant-volume) chamber with sapphire optical windows, and a camera, which can operate in two modes. One mode enables sharp visual observation of the glass transition and other low-temperature physical processes, including cracking, annealing, ice and hydrate crystallization, cavity formation, melting, etc. The other mode enables tracking of the optical temperature-evolution of the system via recorded light intensity, which we demonstrate may be used to measure the onset glass transition temperature with accuracy similar to differential scanning calorimetry (DSC), and to identify the temperature coordinates of other phase change events. The isovitriscope thus offers a single device combining the phenomenological insight of conventional visual inspection with the quantitative insight of techniques like calorimetry, at the >1 mL volume scales increasingly relevant to cryopreservation applications. To demonstrate uses of the isochoric optical platform, we herein conduct a series of observational studies examining the rich multi-phase phenomena at play during isochoric vitrification of binary cryoprotectant solutions; the effect of surface wettabilities on crack formation in the glassy state; the analogy between differential calorimetric and optical analysis; and more. In summary, the isochoric vitrification cryo-macroscope, or isovitriscope, adds a valuable new tool for the study of aqueous vitrification processes.

Responses of Simulated Arctic Mixed-Phase Clouds to Parameterized Ice Particle Shape

Abstract In simulations of Arctic mixed-phase clouds, cloud persistence and the liquid water path (LWP) are sensitive to ice particle number concentrations. Here, we explore sensitivities of cloud microphysical properties to the dominant ice particle shape (dendrites, plates, columns, or spheres) using the SCALE-AMPS large-eddy simulation model. AMPS is a bin microphysics scheme that predicts particle shapes based on the inherent growth ratio (IGR) of spheroids, which determines vapor depositional growth rates along the a and c axes, and the rimed and aggregate mass fractions. We examine the impacts of various IGR values on simulations of clouds observed during the M-PACE and SHEBA experiments. Under M-PACE (SHEBA) conditions, LWP varies between 49 (1.1) and 230 (6.7) g m−2, and the ice water path (IWP) varies between 3 (0.03) and 40 (0.12) g m−2, depending on the ice shape. The lowest LWP and the highest IWP are obtained when columnar particles dominate because their low terminal velocities and large capacitance and collisional area result in large vapor deposition and riming rates, whereas the highest LWP and lowest IWP are obtained when spherical particles dominate because their vapor deposition and riming rates are low. Because ice particle shape significantly influences simulated Arctic mixed-phase clouds, reliable simulations require accurately estimated IGR values under various atmospheric conditions. Finally, comparisons between the simulation results and observations show that the size distribution larger than 2000 μm is better reproduced when the increase in rimed mass that causes ice particles to become spherical is suppressed. Significance Statement Atmospheric models have difficulties in reproducing Arctic mixed-phase clouds because of uncertainties in the parameterization of microphysical processes. This is the first study to use a large-eddy simulation model implemented with a habit-predicting bin microphysics scheme to demonstrate the important role of ice particle shape on the microphysical properties of both heavy-riming and no-riming mixed-phase clouds. We found the vapor deposition and riming rates to be greatly influenced by ice particle shape. By comparing the ice particle size distribution, mass–diameter relationship, and area ratio between simulation results and observations, we show that a hexagonal ice shape model and a riming model that simply converts ice crystals to graupel may not accurately reproduce actual heavy-riming clouds.

Nacre-inspired hierarchical framework enables tough and impact-monitoring epoxy nanocomposites

Multifunctional epoxy nanocomposites hold great promise for artificial structural materials across civil, industrial, and aerospace fields but were severely impeded by catastrophic brittleness under damage and lack of chemical activity resulting from highly cross-linked networks, leading to poor toughness and absent functionality. Herein, we report a facile yet efficient strategy for fabrication of graphene-encapsulated natural rubber frameworks through nanogroove-based freeze-casting technology, leading to highly tough hierarchically biomimetic epoxy nanocomposites. As judiciously elucidated by physicochemical and morphological characterizations, graphene sheets encapsulated natural rubber were bidirectionally aligned through ice crystals expelled from oriented nanogrooved surface to build hierarchical layered architecture with nanoasperities. Moreover, notable crack deflection derived from the “brick-mortar” structure in epoxy composite and nanoasperities debonding at the interlocking “hard-soft-hard” interface dissipate large amount of energy during fracture process, resulting in boosted fracture toughness. Accordingly, a synergistic performance enhancement is achieved, i.e., more than 3.32-fold increase in fracture toughness at low rubber content (4.3 wt%), and efficient real-time impact self-monitoring and evaluation. This work investigated for the first time the role of graphene-encapsulated natural rubber framework with nanoasperities structure in the reinforcement of epoxy nanocomposites. The design approach outlines a way for high-toughness, multifunctional and long-lasting materials for various engineering applications.

Research on the effect of microelement salt with fertilizer

The visual-polythermal method was used to study the solubility of components in the ZnSO4 – (NH4)2SO4 – H2O system over the temperature interval of -22.0°С to +36.1°С. Solubility and characteristics of the constituents of the system at different temperatures and concentrations were studied to show the physicochemical interactions between zinc sulfate and ammonium sulfate. The phase description delineates the areas of ice crystallization, (NH4)2SO4, ZnSO4ꞏ(NH4)2SO4ꞏ6H2O, and ZnSO4. A solubility diagram was drawn and a new compound, ZnSO4ꞏ(NH4)2SO4ꞏ6H2O, was isolated. The system ZnSO4 – (NH4)2SO4 – H2O was investigated using thirteen inner sections. At the double and triple points of the system, the equipoise composition of solutions and crystallization temperatures were clarified. The analysis was carried out using contemporary physicochemical analysis methods to confirm the identity of the obtained compound. Analytical data on physicochemical changes, thermal stability and chemical decomposition of the ZnSO4ꞏ(NH4)2SO4ꞏ6H2O sample with temperature change were presented. All the diffractographic changes in the X-ray pattern of a new compound, in particular, the set of distances between the planes, the activation of their reflection angles, and the diffraction lines confirm the individuality of the new compound.

A comprehensive review on isochoric freezing: a recent technology for preservation of food and non-food items

Isochoric freezing has impeccable applications in the frozen food industry since it preserves food products without inducing ice crystal formation and also increases the marketability of various food products currently unsuitable for traditional freezing methods.

Solubility in manganese sulfate-ammonium sulfate-water system

The production of phosphorus-containing fertilizers involves the use of mineral acids and phosphate raw materials. In the production process and finished fertilizers, the existence of calcium phosphates, ammonium, sulfuric or nitric and phosphoric acids is possible. Enriched superphosphate contains monocalcium phosphate, calcium sulfate dihydrate, phosphoric acid. During ammoniation, monoammonium phosphate can also be formed. The solubility of the components in the manganese sulfate-ammonium sulfate-water system was studied by the visual-polythermal method in the temperature range from -1.0°С to 27.0°С. The phase diagram limits the regions of ice crystallization, ammonium sulfate, manganese sulfate and chemical compound. A solubility diagram was plotted and a new compound (ammonium sulfate + marganes sulfate + water) (NH4)2Mn(SO4)2*6H2O was isolated. The new compound was identified by chemical, X-ray diffraction, thermogravimetric and IR spectroscopic analyses. To physicochemically substantiate the behavior of marganese sulfate under the conditions of production of enriched superphosphate, the interaction of marganese sulfate with monocalcium phosphate was studied by the method of isomolar series in 0.01 M solutions.

Observation Method of Microphysical Parameters of Ice Crystals in Cloud Based on Digital Holography

Observation Method of Microphysical Parameters of Ice Crystals in Cloud Based on Digital Holography

MXene, protein, and KCl-assisted ionic conductive hydrogels with excellent anti-freezing capabilities, self-adhesive, ultra-stretchability, and remarkable mechanical properties for a high-performance wearable flexible sensor.

Developing a hydrogel with switchable features and freeze tolerance is remarkably significant for designing flexible electronics to adjust various application needs. Herein, MXenes, AFPs (antifreeze proteins), and potassium chloride (KCl) were introduced to a polyacrylamide (PAM) polymer network to design an anti-freezing hydrogel. The ionic hydrogels are characterized by excellent ionic conductivity, presenting adjustable properties of remarkable mechanical strength and self-adhesion to meet individualized application demands. The capability of KCl and AFPs to inhibit ice crystals gives hydrogels with anti-icing properties under a low-temperature atmosphere. The PAM/MXene15/AFP30/KCl15 hydrogel demonstrated negligible hysteresis behavior, quick electromechanical response (0.10 s), and excellent sensitivity (gauge factor (GF) = 13.1 within the strain range of 1200-2000%). The resulting hydrogel could be immobilized on the animal or human skin to detect different body movements and physiological motions, offering reproducibility and precise accuracy as primary advantages. The approach of developing materials with tunable features, along with inorganic salt and the fish-inspired freeze-tolerance method, offers a new prospect for wearable gadgets.

Tuning the low-temperature phase behavior of aqueous ionic liquids.

Water's anomalous behavior is often explained using a two-liquid model, where two types of water, high-density liquid (HDL) and low-density liquid (LDL), can be separated via a liquid-liquid phase transition (LLPT) at low temperature. Mixtures of water and the ionic liquid hydrazinium trifluoroacetate were suggested to also show an LLPT but with the advantage that there is no rapid ice crystallization hampering its observation. It remains controversial whether these solutions exhibit an LLPT or are instead associated with complex phase separation phenomena. We here show detailed low-temperature calorimetry and diffraction experiments on aqueous solutions containing hydrazinium trifluoroacetate and other similar ionic liquids, all at a solute mole fraction of x = 0.175. Hydrazinium trifluoroacetate, ammonium trifluoroacetate, ethylammonium trifluoroacetate and hydrazinium pentafluoropropionate all boast exothermic transitions unrelated to crystallization as well as remarkable structural changes upon cooling into the glassy state. We propose a model inspired by micelle formation and decomposition in surfactant solutions, which is complemented by MD simulations and allows rationalizing the rich phase behavior of our mixtures during cooling. The fundamental aspect of the model is the hydrophobic nature of fluorinated anions that enables aggregation, which is reversed upon cooling and culminates in the remarkable exothermic first-order transition observed at low temperature. That is, we assign the first-order transition not to an LLPT but to phase-separations similar to the ones when falling below the Krafft temperature. All other solutions merely show simple vitrification behavior. Still, they exhibit distinct differences in liquid fragility, which is decreased continuously with decreasing hydrophobicity of the anions. This might enable the systematic tuning of ionic liquids with the goal of designing aqueous solutions of specific fragility.

Anhydride esterification to regulate water migration and reduce ice crystal formation in κ-carrageenan gel during freezing

In this study, κ-carrageenan was chemically modified by acid anhydride esterification to produce acid anhydride-esterified κ-carrageenan, and its properties were subsequently determined. Acid anhydride-esterified κ-carrageenan can improve the water retention properties of gels and limit the deterioration of gel textural performances during the freezing process. Scanning electron microscopy results before and after freezing showed that the frozen esterified κ-carrageenans had a more regular and less fibrous network structure, which suggests that the esterified κ-carrageenans better maintained the gel network structure. These phenomena were studied in relation to intermolecular forces, water movement, and ice crystal formation. The intermolecular force analysis showed that the esterification of anhydride disrupted the hydrogen bond formed between κ-carrageenan molecules, enhanced hydrophobic interactions among κ-carrageenan molecules, and resulted in stronger electrostatic repulsion. Low-field nuclear magnetic resonance analysis showed that acid anhydride-esterified κ-carrageenan more effectively bound to water molecules during freezing. Differential scanning calorimetry results showed that acid anhydride-esterified κ-carrageenan reduced the amount of freezing water and lowered the eutectic point of the gel. The results showed that hydrogen bonding, electrostatic interactions, and hydrophobic interactions modified the structure of the acid anhydride-esterified κ-carrageenan gel network and its binding to water molecules. This phenomenon allowed acid anhydride-esterified κ-carrageenan to better maintain binding with water molecules during the freezing process, which in turn delayed and reduced the formation of ice crystals and greatly decreased the effect of ice crystals on the structure of the gel network.

Microbiological, Physicochemical, and Sensory Characters of Synbiotic Ice Cream from Fermented Milk Using Lactiplantibacillus plantarum subsp. Plantarum Dad-13 Combined with Inulin

Ice cream, which includes dairy product, is a good carrier of addition probiotics and prebiotics. This study was designed to assess the microbiological, physicochemical, and sensory properties of the ice cream. The characteristics of ice cream made from fermented milk using L. plantarum Dad-13 combined with inulin (0%, 1%, and 2%) were evaluated such as cell viability, pH, titratable acidity, overrun, melting rate, sensory evaluation with hedonic test, microstructure using scanning electron microscopy, and volatile organic compounds using HS-GC-MS. The results showed that cell viability in synbiotic ice cream with 2% inulin decreased by 1 log cycle, which showed the most stable value during storage until the 12th week was still 1.03 x 107 CFU/g. Synbiotic ice cream with 2% inulin showed the highest overrun value of 35.72% and the slowest melting rate of 40.71% of ice cream melted in 20 minutes. Overall attributes in the hedonic test of synbiotic ice cream with 2% inulin showed a value of 4, which means the most preferred by panelists. Hence, this research showed that ice cream containing 2% inulin, in particular, has a better microstructure due to the prevention of ice recrystallization, which results in smaller ice crystals. The ketone volatile organic compound only found in ice cream with 1% inulin was cyclopentadecanone, 2-hydroxy- with percentage of area 10.25% while for ice cream that contains 2% inulin, it was oxacyclotetradecan-2-one with percentage of area 9.31%. Furthermore, several volatile organic compounds, such as 2-trifluoroacetoxydodecane, 4-propionyloxytridecane, and anthracene, 9-butyltetradecahydro, were only found in the synbiotic ice cream. This study has the potential to be a novel functional food containing probiotic indigenous L. plantarum Dad-13 and prebiotic inulin.

Physics of Ice Nucleation and Antinucleation: Action of Ice-Binding Proteins.

Ice-binding proteins are crucial for the adaptation of various organisms to low temperatures. Some of these, called antifreeze proteins, are usually thought to inhibit growth and/or recrystallization of ice crystals. However, prior to these events, ice must somehow appear in the organism, either coming from outside or forming inside it through the nucleation process. Unlike most other works, our paper is focused on ice nucleation and not on the behavior of the already-nucleated ice, its growth, etc. The nucleation kinetics is studied both theoretically and experimentally. In the theoretical section, special attention is paid to surfaces that bind ice stronger than water and thus can be "ice nucleators", potent or relatively weak; but without them, ice cannot be nucleated in any way in calm water at temperatures above -30 °C. For experimental studies, we used: (i) the ice-binding protein mIBP83, which is a previously constructed mutant of a spruce budworm Choristoneura fumiferana antifreeze protein, and (ii) a hyperactive ice-binding antifreeze protein, RmAFP1, from a longhorn beetle Rhagium mordax. We have shown that RmAFP1 (but not mIBP83) definitely decreased the ice nucleation temperature of water in test tubes (where ice originates at much higher temperatures than in bulk water and thus the process is affected by some ice-nucleating surfaces) and, most importantly, that both of the studied ice-binding proteins significantly decreased the ice nucleation temperature that had been significantly raised in the presence of potent ice nucleators (CuO powder and ice-nucleating bacteria Pseudomonas syringae). Additional experiments on human cells have shown that mIBP83 is concentrated in some cell regions of the cooled cells. Thus, the ice-binding protein interacts not only with ice, but also with other sites that act or potentially may act as ice nucleators. Such ice-preventing interaction may be the crucial biological task of ice-binding proteins.

Cold climatic snaps during the Eocene-Oligocene transition in the central Tibetan Plateau: Implications for ice-induced sedimentary structures and isotope geochemistry

The Eocene-Oligocene transition was a time of major climate instability and a shift from an ice-free world to a predominant icehouse climate. Identifying the ancient glacial deposits and their associated sedimentary structures are of paramount significance for understanding the Earth's climate in the Cryogenian Period. Ice-induced sedimentary structures (IISS) are considered as types of primary structures preserved on surfaces and in upper parts of unconsolidated sediments by physical cryospheric processes of seasonal ice freezing and thawing, and therefore sensitive not only to climate, but also to paleogeography, hydrology, and type of environment. Despite the common occurrence of modern IISS in a wide variety of environments, from high-latitude regions to low-latitude areas of high altitude, available records from ancient geologic archives are scarce. Herein, we integrated sedimentological and geochemical proxies to study IISS and geochemical signals as reliable indicators of cold climate in an outstanding example of the Eocene-Oligocene interval from the Niubao Formation in the Lunpola Basin, central Tibet. Detailed field sedimentological, petrographic microscopic investigations, and bulk carbonate oxygen (δ18Ocarb) and carbon (δ13Ccarb) isotope geochemistry were conducted in this lacustrine succession. Seven sets of IISS are represented by ice crystal marks, frozen cracks, ice-frozen bubbles, ice-expanding laminae, ice-rafted debris (dropstones), ice water pea-like, and ice water pits. They are classified into three groups based on major deriving mechanisms: ice-related crystal marks, deformation-related ice structures, and gravitational fall IISS. Positive δ18Ocarb excursions at the Eocene-Oligocene transition provide evidence of a shift toward cold or near-freezing climatic snaps in response to global cooling episodes at this time. Integrating the paleogeomorphic scenario of central Tibet with the stratigraphically reported IISS and glendonite clusters, the results support that the Lunpola lake sustained cold or near-freezing conditions at Eocene-Oligocene transition. These findings provide one of the most comprehensive sedimentological and isotopic analyses of ancient IISS and a more direct spatial-temporal constraint for the regional climate system of central Tibet.

Key factors for the survival of Lactiplantibacillus plantarum IDCC 3501 in manufacturing and storage.

Functional microbiome development has steadily increased; with this, the viability of microbial strains must be maintained not only after the manufacturing process but also at the time of consumption. Survival is threatened by various unavoidable factors during freeze-drying and shelf storage. Here, the aim was to optimize the manufacturing process of the functional strain Lactiplantibacillus plantarum IDCC 3501 after freeze-drying and storage. Explosive growth was achieved using a medium composition with two nitrogen sources and a mineral, and growth was drastically improved by neutralizing the medium pH during the culture of L. plantarum IDCC 3501. Culture optimization involved a smaller cell size, leading to less intracellular free water. Moreover, when maltodextrin (MD) powder was directly added to the harvested cells, some intracellular free water was extracted from the bacterial cells, resulting in a dramatic increase in the viability of L. plantarum IDCC 3501 after freeze-drying and subsequent storage. Furthermore, MD enhanced survival in a dose-dependent manner. Bacterial survival was correlated with lysozyme tolerance; therefore, the positive result might have been caused by the osmotic dehydration of intracellular free water, which would potentially damage the bacterial cells via ice crystallization and/or a phase transition during freeze-drying. These critical factors of L. plantarum IDCC 3501 processing provide perspectives on survival issues for manufacturing microbiome strains. KEY POINTS: • Culture conditions for probiotic bacteria were optimized for high growth yield. • Osmotic dehydration improved bacterial survival after manufacturing and shelf storage. • Reduction in intracellular free water content is crucial for intact survival.