Glassy State and Seed Storage Stability: A Viability Equation Analysis

Dry seeds exist generally in a glassy (or vitrified) state.The high viscosity of the glassy state would be expected tohave a retarding effect on deteriorative reactions in the cytoplasm.Thus the glassy state may be considered to be a biophysicalbarrier for seed deterioration. The present study aims to testthe hypothesis that seed storage stability is associated withthe glassy state. With the equations derived from the seed viabilityequation, we have calculated the maximum temperature (Tmax)for long-term storage of corn, pea and soybeans. The Tmax forlong-term seed storage is found to be in a good agreement withthe glass transition temperature (Tg) in each instance, suggestingthat seed deterioration would be accelerated when seeds arenot in the glassy state. Experiments with soybeans given acceleratedageing show that the loss of glassy state is followed by a rapiddecrease in seed viability. These observations provide indirectevidence that the glassy state may play a significant role inseed storage stability.Copyright 1994, 1999 Academic Press Glassy state, seed longevity, storage stability, viability analysis     

Protein stability in the amorphous carbohydrate matrix: relevance to anhydrobiosis

The formation of intracellular glass is proposed to be relevant to protein stabilization and survival of anhydrobiotic organisms in the dry state. The stability of proteins in the amorphous carbohydrate matrix and its relevance to seed survival have been investigated in the present study. Glucose-6-phosphate dehydrogenase (G6PDH) was preserved in the amorphous glucose/sucrose (1:10, w/w) matrix by freeze-drying. The stability of freeze-dried G6PDH was examined at temperatures above and below the glass transition temperature (T_g). The rate of G6PDH inactivation in the amorphous carbohydrate matrix deviated significantly from the Arrhenius kinetics, and conformed to the Williams-Landel-Ferry (WLF) relationship. The temperature dependence of G6PDH inactivation in two sets of samples with different T_g values was compared. Identical temperature dependence of G6PDH inactivation was observed after temperature normalization by (T?T_g). Seed survival of Vigna radiata Wilczek (mung bean) showed a similar WLF kinetics at storage temperatures T≥T_g. In situ protein stability in mung bean embryonic axes was studied using differential scanning calorimetry (DSC). Thermal stability of seed proteins exhibited a strong dependence on the T_g of intracellular glass. These results indicate an important role of the glassy state in protein stabilization. Our data suggest an association between protein stability in intracellular glass and seed survival during storage.     

A Calorimetric Study of the Glass Transition Behaviors in Axes of Bean Seeds with Relevance to Storage Stability

Although the presence of intracellular aqueous glasses has been established in seeds, their physiological role in storage stability is still conjectural. Therefore, we examined, using differential scanning calorimetry, the thermal behavior of glass transitions in axes of bean (Phaseolus vulgaris L.) with water contents (WC) between 0 and 1 g H2O/g dry weight (g/g) and temperatures between -120 and +120[deg]C. Three types of thermal behaviors associated with the glass transition were observed. The appearance, the glass -> liquid transition temperature, and the amount of energy released during these transitions were dependent on the tissue WC. No glass transitions were observed at WC lower than 0.03 and higher than 0.45 g/g. A brief exposure to 100[deg]C altered the glass properties of tissues with WC between 0.03 and 0.08 g/g but did not affect the thermal behavior of glasses with higher WC, demonstrating that thermal history is important to the intracellular glass behavior at lower WC. Correspondence of data from bean to models predicting the effects of glass components on the glass -> liquid transition temperature suggests that the intracellular glasses are composed of a highly complex sugar matrix, in which sugar and water molecules interact together and influence the glass properties. Our data provide evidence that additional glass properties must be characterized to understand the implications of a glassy state in storage stability of seeds.     

Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation,sugar hydrolysis,Maillard reactions and their relationship to glass state transition

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.     

The amorphous state of spray-dried maltodextrin: sub-sub-Tg enthalpy relaxation and impact of temperature and water annealing

The annealing behaviour of a spray-dried maltodextrin was investigated by differential scanning calorimetry. Special attention was paid to the effect of temperature and humidity on the annealing process. Comparison was also made with the glassy state of the same compound prepared by various cooling processes. The presence of a very pronounced sub-T_g peak upon ageing reveals the specificities of the glass and the complexity of the relaxation spectrum of the spray-dried material. This peak seems actually to correspond to a partial ergodicity recovery that may be attributed to onset of molecular mobility occurring below T_g. The position of the sub-T_g peak with regard to the conventional T_g was systematically studied. It clearly showed the difference between the effect of temperature and water plasticization on the relaxations occurring in the glassy state of materials prepared by spray-drying.     

Evaluation of the relevance of the glassy state as stability criterion for freeze-dried bacteria by application of the Arrhenius and WLF model

The aim of this work was to describe the temperature dependence of microbial inactivation for several storage conditions and protective systems (lactose, trehalose and dextran) in relation to the physical state of the sample, i.e. the glassy or non-glassy state. The resulting inactivation rates k were described by applying two models, Arrhenius and Williams–Landel–Ferry (WLF), in order to evaluate the relevance of diffusional limitation as a protective mechanism. The application of the Arrhenius model revealed a significant decrease in activation energy E_a for storage conditions close to T_g. This finding is an indication that the protective effect of a surrounding glassy matrix can, at least, partly be ascribed to its inherent restricted diffusion and mobility. The application of the WLF model revealed that the temperature dependence of microbial inactivation above T_g is significantly weaker than predicted by the universal coefficients. Thus, it can be concluded that microbial inactivation is not directly linked with the mechanical relaxation behavior of the surrounding matrix as it was reported for viscosity and crystallization phenomena in case of disaccharide systems.     

Stability of biocontrol products carrying Candida sake CPA-1 in starch derivatives as a function of water activity

The preservation and shelf-life of formulations of the biocontrol agent Candida sake CPA-1 and starch derivatives as a function of water activity (a_W) were studied in terms of the physical stability of the products and cell viability. Formulations of biocontrol products (BCPs), based on combinations of potato starch and pre-gelatinised potato starch (F1 and F2) or maltodextrines (MD) (F3) containing cell protectants, were obtained by fluidised-bed drying. The carriers and the formulated products were stored at 20°C under different a_W conditions. The water sorption and water plasticization behaviour of the different products were analysed through the water sorption isotherms and glass transition temperatures (T_g). Likewise, the viability of C. sake over time was determined as a function of the a_W. The solubility of the products was also assessed. Although formulations stored at 20°C and low a_W (≤?0.33) exhibited a better shelf-life, a significant decrease in cell survival ratio after 180 storage days was observed. Cold storage (5°C) was required to better maintain the cell viability, thus prolonging the shelf-life of BCPs. Formulations containing MD were the most effective at preserving cell viability and also exhibited the highest water solubility. All the formulations were physically stable at ambient temperature; therefore, the cell stability is the critical point at which to establish both the a_W levels and temperature during storage. Packaging the product using high water vapour barrier material and under cold storage would be necessary to ensure a high number of viable cells and an effective and competitive BCP.     

Intracellular glasses and seed survival in the dry state

So-called orthodox seeds can resist complete desiccation and survive the dry state for extended periods of time. During drying, the cellular viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. The formation of intracellular glasses is indispensable to survive the dry state. Indeed, the storage stability of seeds is related to the packing density and molecular mobility of the intracellular glass, suggesting that the physico-chemical properties of intracellular glasses provide stability for long-term survival. Whereas seeds contain large amounts of soluble non-reducing sugars, which are known to be good glass formers, detailed in vivo measurements using techniques such as FTIR and EPR spectroscopy reveal that these intracellular glasses have properties that are quite different from those of simple sugar glasses. Intracellular glasses exhibit slow molecular mobility and a high molecular packing, resembling glasses made of mixtures of sugars with proteins, which potentially interact with additional cytoplasmic components such as salts, organic acids and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a low molecular mobility and a high stability, which serves as an ecological advantage, keeping the seeds stable under adverse conditions of temperature or water content that bring the tissues out of the glassy state.     

Evaluation of the relevance of the glassy state as stability criterion for freeze-dried bacteria by application of the Arrhenius and WLF model

The aim of this work was to describe the temperature dependence of microbial inactivation for several storage conditions and protective systems (lactose, trehalose and dextran) in relation to the physical state of the sample, i.e. the glassy or non-glassy state. The resulting inactivation rates k were described by applying two models, Arrhenius and Williams–Landel–Ferry (WLF), in order to evaluate the relevance of diffusional limitation as a protective mechanism. The application of the Arrhenius model revealed a significant decrease in activation energy E_a for storage conditions close to T_g. This finding is an indication that the protective effect of a surrounding glassy matrix can, at least, partly be ascribed to its inherent restricted diffusion and mobility. The application of the WLF model revealed that the temperature dependence of microbial inactivation above T_g is significantly weaker than predicted by the universal coefficients. Thus, it can be concluded that microbial inactivation is not directly linked with the mechanical relaxation behavior of the surrounding matrix as it was reported for viscosity and crystallization phenomena in case of disaccharide systems.     

含水量对种子贮藏寿命的影响

建立以收集种子为主体的基因库乃是当今保护植物种质资源最为普遍且可靠易行的方式,在世界库存约 61 0 0 0 0 0份种质资源中,近 90 %是以种子形式保存于约 1 30 0个基因库中。低温贮藏仍是目前基因库中种子种质保存的主要方法。种子含水量和贮藏温度是影响种子在贮藏期间生活力和活力保持的关键因素。传统的经验认为控制温度比控制水分来得安全有效,因而趋向于向低温或超低温的贮藏方向发展。国际植物遗传资源研究所（ＩＰＧＲＩ）曾推荐 5%～ 6%的含水量和 - 1 8～- 2 0℃低温作为各国长期保存种子的理想条件。目前,世界各国都把更多的…     

Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.     

Glass formation in plant anhydrobiotes: survival in the dry state

Anhydrobiotes can resist complete dehydration and survive the dry state for extended periods of time. During drying, cytoplasmic viscosity increases dramatically and in the dry state, the cytoplasm transforms into a glassy state. Plant anhydrobiotes possess large amounts of soluble non-reducing sugars and their state diagrams resemble those of simple sugar mixtures. However, more detailed in vivo measurements using techniques such as Fourier transform infrared spectroscopy and electron paramagnetic resonance spectroscopy reveal that these intracellular glasses are complex systems with properties quite different from those of simple sugar glasses. Intracellular glasses exhibit a high molecular packing and slow molecular mobility, resembling glasses made of mixtures of proteins and sugars, which potentially interact with additional cytoplasmic components such as salts, organic acids, and amino acids. Above the glass transition temperature, the cytoplasm of biological systems still exhibits a high stability and low molecular mobility, which could serve as an ecological advantage. All desiccation-tolerant organisms form glasses upon drying, but desiccation-sensitive organisms generally lose their viability during drying at water contents at which the glassy state has not yet been formed, suggesting that other factors are necessary for desiccation tolerance. Nevertheless, the formation of intracellular glasses is indispensable to survive the dry state. Storage stability of seeds and pollens is related to the molecular mobility and packing density of the intracellular glass, suggesting that the characteristic properties of intracellular glasses provide stability for long-term survival.     

Understanding the Influence of State/Phase Transitions on Ice Recrystallization in Atlantic Salmon (Salmo salar) During Frozen Storage

Temperature fluctuations during storage and distribution of frozen foods lead to ice recrystallization and microstructural modifications that can affect food quality. Low temperature transitions may occur in frozen foods due to temperature fluctuations, resulting in less viscous and partially melted food matrices. This study systematically investigated the influence of state/phase transitions and temperature fluctuations on ice recrystallization during the frozen storage of salmon fillets. Using a modulated differential scanning calorimeter, we identified the characteristics glass transition temperature (T _g ′) of −27 °C and the onset temperature for ice crystal melting (T _m ′) of −17 °C in salmon. The temperature of salmon fillets in sealed plastic trays was lowered to −35 °C in a freezer to achieve the glassy state. The temperature (T) of frozen salmon fillets in sealed plastic trays was modulated to achieve a rubbery state (T > T _m ′), a partially freeze-concentrated state (T _g ′ < T < T _m ′) and a glassy state (T < T _g ′). We performed temperature modulation experiments by exposing packaged salmon to room temperature twice a day for 2 to 26 min during 4 weeks of storage. We also analyzed ice crystal morphology using environmental scanning electron microscopy and X-ray computed tomography techniques to observe the pore distribution after sublimation of ice crystals. Melt–refreeze and isomass rounding mechanisms of ice recrystallization were noticed in the frozen salmon subjected to temperature modulations. Results show that ice crystal growth occurred even in the glassy state of frozen salmon during storage, with or without temperature fluctuations. Ice crystal size in frozen salmon was greater in the rubbery state (T > T _m ′) due to the increased mobility of unfrozen water compared to the glassy state. The morphological/geometric parameters of ice crystals in frozen salmon stored for 1 month differed significantly from those in 0-day storage. These findings are important to the frozen food industry because they can help optimize storage and distribution conditions and minimize quality loss of frozen salmon due to recrystallization.     

Physicochemical Investigations and Stability Studies of Amorphous Gliclazide

Gliclazide (GLI), a poorly water-soluble antidiabetic, was transformed into a glassy state by melt quench technique in order to improve its physicochemical properties. Chemical stability of GLI during formation of glass was assessed by monitoring thin-layer chromatography, and an existence of amorphous form was confirmed by differential scanning calorimetry and X-ray powder diffractometry. The glass transition occurred at 67.5°C. The amorphous material thus generated was examined for its in vitro dissolution performance in phosphate buffer (pH 6.8). Surprisingly, amorphous GLI did not perform well and was unable to improve the dissolution characteristics compared to pure drug over entire period of dissolution studies. These unexpected results might be due to the formation of a cohesive supercooled liquid state and structural relaxation of amorphous form toward the supercooled liquid region which indicated functional inability of amorphous GLI from stability point of view. Hence, stabilization of amorphous GLI was attempted by elevation of T_g via formation of solid dispersion systems involving comprehensive antiplasticizing as well as surface adsorption mechanisms. The binary and ternary amorphous dispersions prepared with polyvinylpyrrolidone K30 (as antiplasticizer for elevation of T_g) and Aerosil 200® and/or Sylysia® 350 (as adsorbent) in the ratio of 1:1:1 (w/w) using kneading and spray-drying techniques demonstrated significant enhancement in rate and extent of dissolution of drug initially. During accelerated stability studies, ternary systems showed no significant reduction in drug dissolution performance over a period of 3 months indicating excellent stabilization of amorphous GLI.Key words: amorphous, gliclazide, solid dispersion, stability studies, T_g     

Theoretical Basis of Protocols for Seed Storage III. Optimum Moisture Contents for Pea Seeds Stored at Different Temperatures

In previous work, we demonstrated that there was an optimummoisture level for seed storage at a given temperature (Vertucciand Roos, 1990), and suggested, using thermodynamic considerations,that the optimum moisture content increased as the storage temperaturedecreased (Vertucci and Roos, 1993b). In this paper, we presentdata from a two year study of aging rates in pea (Pisum sativum)seeds supporting the hypothesis that the optimum moisture contentfor storage varies with temperature. Seed viability and vigourwere monitored during storage under dark or lighted conditionsat relative humidities between 1 and 90%, and temperatures between-5 and 65°C. The optimum moisture content varied from 0·015g H₂O g^-1 d.wt at 65°C to 0·101 g H₂O g^-1 d.wt at15°C under dark conditions and from 0·057 at 35°Cto 0·092 g H₂O g^-1 d.wt at -5°C under lighted conditions.Our results suggest that optimum moisture contents cannot beconsidered independently of temperature. This conclusion hasimportant implications for 'ultra-dry' and cryopreservationtechnologies.Copyright 1994, 1999 Academic Press Seed storage, seed aging, seed longevity, water content, temperature, glass, desiccation damage, ultradry, Pisum sativum L., pea, cryopreservation     

Wheat seed ageing viewed through the cellular redox environment and changes in pH

To elucidate biochemical mechanisms leading to seed deterioration, we studied 23 wheat genotypes after exposure to seed bank storage for 6–16 years compared to controlled deterioration (CD) at 45?°C and 14 (CD14) and 18% (CD18) moisture content (MC) for up to 32 days. Under two seed bank storage conditions, seed viability was maintained in cold storage (CS) at 0?°C and 9% seed MC, but significantly decreased in ambient storage (AS) at 20?°C and 9% MC. Under AS and CS, organic free radicals, most likely semiquinones, accumulated, detected by electron paramagnetic resonance, while the antioxidant glutathione (GSH) was partly lost and partly converted to glutathione disulphide (GSSG), detected by HPLC. Under AS the glutathione half-cell reduction potential (E_GSSG/2GSH) shifted towards more oxidising conditions, from ?186 to ?141?mV. In seeds exposed to CD14 or CD18, no accumulation of organic free radicals was observed, GSH and seed viability declined within 32 and 7 days, respectively, GSSG hardly changed (CD14) or decreased (CD18) and E_GSSG/2GSH shifted to ?116?mV. The pH of extracts prepared from seeds subjected to CS, AS and CD14 decreased with viability, and remained high under CD18. Across all treatments, E_GSSG/2GSH correlated significantly with seed viability (r?=?0.8, p<.001). Data are discussed with a view that the cytoplasm is in a glassy state in CS and AS, but during the CD treatments, underwent transition to a liquid state. We suggest that enzymes can be active during CD but not under the seed bank conditions tested. However, upon CD, enzyme-based repair processes were apparently outweighed by deteriorative reactions. We conclude that seed ageing by CD and under seed bank conditions are accompanied by different biochemical reactions.     

Water activity‐temperature state diagrams of freeze‐dried Lactobacillus acidophilus (La‐5): Influence of physical state on bacterial survival during storage

Water activity‐temperature state diagrams for Lactobacillus acidophilus freeze‐dried in a sucrose or a lactose matrix were established based on determination of stabilized glass transition temperatures by differential scanning calorimetry during equilibration with respect to water activity at fixed temperatures. The bacteria in the lactose matrix had higher stabilized glass transition temperatures for all a_w investigated. The survival of Lactobacillus acidophilus determined as colony forming units for up to 10 weeks of storage at 20°C for (i) a_w = 0.11 with both freeze‐dried matrices in the glassy state, (ii) a_w = 0.23 with the bacteria in the lactose matrix in a glassy state but with the bacteria in sucrose matrix in the nonglassy state, and (iii) a_w = 0.43 with both freeze‐dried matrices in a nonglassy state showed that the nature of the sugar was more important for storage stability than the physical state of the matrix with the nonreducing sucrose providing better stability than the reducing lactose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Seed germination requirements of Trembleya laniflora (Melastomataceae), an endemic species from neotropical montane rocky savannas

Trembleya laniflora is an endemic shrub from neotropical montane rocky savannas of southeastern Brazil. It has been indicated as a potential candidate for ecological restoration of abandoned iron‐ore mines due to heavy metal accumulation. Here, we evaluated the seed germination requirements of T. laniflora. Seeds collected in 2005 and 2008 were set to germinate under a broad range of temperature and light conditions. Seed viability was estimated by dissecting seeds under a dissecting microscope for embryo presence/absence. Seeds were photoblastic and optimum temperature range was 20–25°C, coinciding with the onset of the rainy season. Seeds were viable after 42 months of storage, which together with small seeds that easily get buried and light requirement for germination suggest formation of soil seed banks. Except the large fraction of embryoless seeds, almost all tested seeds germinated when incubated under light conditions; therefore, T. laniflora should be regarded to have nondormant seeds. Easiness of burial resulting from small seed size and positive photoblastism may both contribute to incorporation into soil seed banks. Our data suggest that the long‐term storage of T. laniflora seeds provides a useful strategy for plant reintroduction.     

Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze‐dried in sucrose

Aims: To investigate the effect of freeze‐dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose‐based formulations and to examine the relationship between amorphous matrix stability and cell viability. Methods and Results: The resistance to moisture‐induced sucrose crystallization and effects on the glass transition temperature (T_g) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. Conclusions: Polymers can be used to enhance the stability of freeze‐dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50° below its T_g for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. Significance and Impact of the Study: The results will aid in the rational formulation design and proper determination of storage conditions for freeze‐dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.