Influence of ISPs from a polar sea-ice microalga on whipped cream detected by cryo-Raman microscopy

The occurrence of recrystallization and large ice areas after storage of frozen food products makes the use of ice-structuring proteins (ISPs) in food products meaningful. Food products are frozen to extend shelf-life during long storage periods while preservation of the overall sensoric quality. Recrystallisation processes alter and, in the worst case, damage the structure of the food, resulting in an unsightly thawed e.g. cream cake or cream puff product. Freezing and freeze storage behaviour of various food products should be investigated in order to optimize the manufacturing of frozen products and to maintain the product quality with appropriate storage until the consumers usage. We present a study on the influence of ice-structuring proteins, isolated from the sea-ice microalgae Fragilariopsis cylindrus (fcISP), on frozen whipped cream. The individual phases of the frozen cream foam have been detected by cryo-Raman spectroscopy and visualized. The advantage of the unique cryo-Raman spectroscopy system available at the AWI is that the individual components can be detected not only qualitatively, but also localized in the frozen sample. We show that the fat and ice structure in frozen cream, and their temperature-induced changes, are well detectable by cryo-Raman spectroscopy. Furthermore, the effect of fcISPs on the microstructure shows an inhibition of ice recrystallization, leading to smaller grain aggregates and a finer fat distribution than without fcISPs. We therefore suggest that fcISPs are an effective mean in controlling recrystallization processes in frozen goods

Growth suppression of ice crystal basal face in the presence of a moderate ice-binding protein does not confer hyperactivity

Ice-binding proteins (IBPs) affect ice crystal growth by attaching to crystal faces. We present the effects on the growth of an ice single crystal caused by an ice-binding protein from the sea ice microalga Fragilariopsis cylindrus (fcIBP) that is characterized by the widespread domain of unknown function 3494 (DUF3494) and known to cause a moderate freezing point depression (below 1 °C). By the application of interferometry, bright-field microscopy, and fluorescence microscopy, we observed that the fcIBP attaches to the basal faces of ice crystals, thereby inhibiting their growth in the c direction and resulting in an increase in the effective supercooling with increasing fcIBP concentration. In addition, we observed that the fcIBP attaches to prism faces and inhibits their growth. In the event that the effective supercooling is small and crystals are faceted, this process causes an emergence of prism faces and suppresses crystal growth in the a direction. When the effective supercooling is large and ice crystals have developed into a dendritic shape, the suppression of prism face growth results in thinner dendrite branches, and growth in the a direction is accelerated due to enhanced latent heat dissipation. Our observations clearly indicate that the fcIBP occupies a separate position in the classification of IBPs due to the fact that it suppresses the growth of basal faces, despite its moderate freezing point depression

Multiple binding modes of a moderate ice-binding protein from a polar microalga

Ice-binding proteins (IBPs) produced by cold-tolerant organisms interact with ice and strongly control crystal growth. The molecular basis for the different magnitudes of activity displayed by various IBPs (moderate and hyperactive) has not yet been clarified. Previous studies questioned whether the moderate activity of some IBPs relies on their weaker binding modus to the ice surface, compared to hyperactive IBPs, rather than relying on binding only to selected faces of the ice crystal. We present the structure of one moderate IBP from the sea-ice diatom Fragilariopsis cylindrus (fcIBP) as determined by X-ray crystallography and investigate the protein's binding modes to the growing ice-water interface using molecular dynamics simulations. The structure of fcIBP is the IBP-1 fold, defined by a discontinuous β-solenoid delimitated by three faces (A, B and C-faces) and braced by an α-helix. The fcIBP structure shows capping loops on both N- and C-terminal parts of the solenoid. We show that the protein adsorbs on both the prism and the basal faces of ice crystals, confirming experimental results. The fcIBP binds irreversibly to the prism face using the loop between the B and the C-faces, involving also the B-face in water immobilization despite its irregular structure. The α-helix attaches the protein to the basal face with a partly reversible modus. Our results suggest that fcIBP has a looser attachment to ice and that this weaker binding modus is the basis to explain the moderate activity of fcIBP

Location and distribution of micro-inclusions in the EDML and NEEM ice cores using optical microscopy and in situ Raman spectroscopy

Impurities control a variety of physical properties of polar ice. Their impact can be observed at all scales – from the microstructure (e.g., grain size and orientation) to the ice sheet flow behavior (e.g., borehole tilting and closure). Most impurities in ice form micrometer-sized inclusions. It has been suggested that these μ inclusions control the grain size of polycrystalline ice by pinning of grain boundaries (Zener pinning), which should be reflected in their distribution with respect to the grain boundary network. We used an optical microscope to generate high-resolution large-scale maps (3 μm pix^-1, 8 x 2 cm^2) of the distribution of micro-inclusions in four polar ice samples: two from Antarctica (EDML, MIS 5.5) and two from Greenland (NEEM, Holocene). The in situ positions of more than 5000 μ inclusions have been determined. A Raman microscope was used to confirm the extrinsic nature of a sample proportion of the mapped inclusions. A superposition of the 2-D grain boundary network and μ-inclusion distributions shows no significant correlations between grain boundaries and μ inclusions. In particular, no signs of grain boundaries harvesting μ inclusions could be found and no evidence of μ inclusions inhibiting grain boundary migration by slow-mode pinning could be detected. Consequences for our understanding of the impurity effect on ice microstructure and rheology are discussed

Location and composition of micro-inclusions in deep ice from the EDML ice core (Antarctica) using optical microscope and cryo-Raman spectroscopy

The impurity content in meteoric ice from polar regions is relatively low compared to other natural materials. However, it controls a variety of physical properties of ice - from dielectric response to its mechanical behaviour. Links between impurity concentration, changes in ice micro-structure and deformation rate have been reported on several scales. In order to approach the responsible mechanisms, a better understanding is needed regarding the in-situ form, location, and distribution of the different species within the polycrystal. We used an optical microscope to generate high-resolution 2D-maps of micro-inclusions in deep ice from the EDML ice core (Antarctica). Superposition of the grain boundary network and micro-inclusion distributions shows no significant correlations between grain boundaries and micro-inclusions. Implications for the relevance of Zener pinning during grain boundary migration and redistribution of impurities by grain boundary drag are discussed. Raman spectra of micro-inclusions in selected regions were obtained using a confocal cryo-Raman system. Comparison with ion chromatography shows that most of the available ions in ice precipitate in form of micro-inclusions. However, indications were found that some of the residual components could coexist in form of solid solution

Ice‐binding proteins and the ‘domain of unknown function’ 3494 family

Ice‐binding proteins (IBPs) control the growth and shape of ice crystals to cope with subzero temperatures in psychrophilic and freeze‐tolerant organisms. Recently, numerous proteins containing the domain of unknown function (DUF) 3494 were found to bind ice crystals and, hence, are classified as IBPs. DUF3494 IBPs constitute today the most widespread of the known IBP families. They can be found in different organisms including bacteria, yeasts and microalgae, supporting the hypothesis of horizontal transfer of its gene. Although the 3D structure is always a discontinuous β‐solenoid with a triangular cross‐section and an adjacent alpha‐helix, DUF3494 IBPs present very diverse activities in terms of the magnitude of their thermal hysteresis and inhibition of ice recrystallization. The proteins are secreted into the environments around the host cells or are anchored on their cell membranes. This review covers several aspects of this new class of IBPs, which promise to leave their mark on several research fields including structural biology, protein biochemistry and cryobiology

Physical properties of the NEGIS ice core - The upper 1700m in EGRIP

We will present the EGRIP CPO (c-axes fabric) dataset and give preliminary interpretations concerning the processes leading to its evolution. 120 bags were selected, with a minimum depth resolution of 15m. Bags were mostly measured continuously, and in total 778 thin sections were prepared, measured and pre-processed on site. Thus, c-axes distribution CPO data are already available, while other parameters on grain stereology are still to be processed at this stage. The CPO patterns found in the upper 1650m at EGRIP show (1) a rapid evolution of c-axes anisotropy compared to lower dynamics sites and (2) partly novel characteristics in the CPO patterns. (1) Starting the measurements at 118m of depth we find a very broad single maximum distribution. The c-axes align with depth in the upper 400m much more rapidly than seen in ice cores from divides or domes. Down to only 140m depth the almost random CPO develops into a very broad single maximum which is similar to those CPOs found in the shallowest samples of other ice cores. Possible interpretations of these distributions are deformation by vertical compression from overlying layers, or alternatively a temperature-gradient snow metamorphosis. This weak CPO pattern is, however, quickly overprinted in the depth zone below 140m where a progressive evolution towards a vertical girdle distribution is observed. As vertical girdles are produced by extension along flow, the observed distribution indicates that the ice at this depth is deforming rather than just being translated by rigid block movement. From approximately 600m of depth downward we observe crystal orientation anisotropy of a strength comparable to samples from ~1400m of depth at divides (NEEM and EDML). This strong girdle CPO remains rather stable down to approximately 1300m depth, where we reach the ice deposited during the last glacial period. A novel pattern, not observed before in natural ice, is a higher densities of c-axes horizontally oriented within the vertical girdle. (2) The early onset of deformation seems further supported by the observation of a broad “hourglass shaped” girdle, which seems to develop in some depths into a “butterfly shaped” cross girdle. Another characteristic deserves attention: the distribution density within the girdle. In contrast to observations in deep ice cores so far, the highest density seems to deviate from the vertical direction being (sub-)parallel to the horizontal. The origin of this may lay in the main deformation modes, e.g. a combination of along flow extension with additional deformation modes. Especially interesting is the cross girdle, which has not yet been observed in polar ice cores so far. We suggest three possible interpretations for its origin: a) In other materials, such as quartz, cross girdles can be interpreted as activation of multiple dislocation slip systems. b) Alternatively, the CPO pattern may reflect reminiscent features from previous deformation modes, which the ice experienced upstream or possibly even outside of the ice stream. This memory effect would point to a relevance of strain dependence of the CPO. c) The cross- /double-girdle might be caused by the early onset of dynamic migration recrystallization under horizontal uniaxial extension

An analysis of the influence of deformation and recrystallisation on microstructures of the EastGRIP ice core

New and more detailed investigations from the EGRIP physical properties dataset down to 1650m of the ice core will be presented. EGRIP is the first deep ice core through one of our Earth’s ice sheets partly motivated by ice dynamics’ research. It is drilled just downstream of the onset of the largest ice stream in Greenland (North East Greenland Ice Stream). Data processing of the collected ice core physical properties data was done at the Alfred Wegener Institute Helmholtz Centre for Marine and Polar Research. The two main findings regarding CPO (c-axes fabric) pattern, 1) a rapid evolution of c-axes anisotropy and 2) partly novel characteristics, were further, and in more detail, investigated. To gain a better understanding of the dominating deformation mechanisms of NEGIS, different approaches considering different length scales were chosen (1650m versus 0.55m and 0.09m scale), including several case studies. A large-scale statistical analysis of the entire dataset results in new information about the depth-dependent evolution of parameters as for example the strength of c-axes anisotropy and grain-size in the polycrystal. In general, mean grain-size decreases with depth as we drill through the Holocene ice and approach the Glacial material. The grain size variability with fine and coarse grain layers is extreme in the Holocene ice but decreases in the Glacial ice. Microstructure properties were examined, with the aim to investigate the relationship between the remarkable rapid evolution of CPO-pattern and grain properties evolution. Furthermore, the evolution of a grain-size dependent anisotropy, found in the first 350m of the ice core, is investigated and examined also in deeper sections of the core. The large-scale evolution of density distributions of c-axes orientations differ significantly from observations in deep ice cores made so far: A novel "hourglass shaped" girdle was observed, characterized by a high density of horizontally oriented c-axes within the vertical girdle. In some parts of the core, this shape develops into a "butterfly shaped" cross girdle, varying in intensity and strength. It is the first time that this cross girdle was observed in polar ice and by combining approaches considering different length scales, we aimed to verify one of our three hypotheses for its origin: a) activation of multiple dislocation slip systems (in analogy to quartz), b) a memory effect or reminiscent features from older deformation modes, further upstream or even outside of NEGIS or c) horizontal uniaxial extension causing an early onset of dynamic migration recrystallization. Small-scale high-resolution studies were carried out on several selected bags (0.55m long) from different depth regimes and were chosen as case studies to better understand the formation mechanisms of the novel CPO patterns found in the EGRIP ice core. One approach is the examination of thin sections (9 x 7 x 0.03cm) regarding the occurrence of patches of grains with similar orientations, which was observed in several samples from different depths. Small grains with similar orientations seem to cluster around large grains with a different orientation. High-resolution images (5 to 20µm/pix), derived with a Large Area Scan Macroscope (LASM), enable detailed investigations of grain shape, grain boundaries and sub-grain boundaries and therefore the possibility to find distinct features from deformation and recrystallisation in the microstructure. Grains are rarely horizontally aligned and usually show irregular, circular or rectangular shapes rather than elongated shapes. Characteristic for our case studies are also amoeboid grain shapes and sutured grain boundaries, typical features of grain boundary migration. Furthermore, layering, "sandwiched grains" and strong gradients in grain-size over distances of only a few centimetres were observed in several samples. Although still under progress, at the current state of investigation, combining fabric data and microstructure analysis, the novel CPO patterns found in the EGRIP ice core are strongly influenced by dynamic migration recrystallisation