Cryoprotective Activity Research Articles

Investigation of the physiochemical properties, cryoprotective activity and possible action mechanisms of sericin peptides derived from membrane separation

In order to exploit their industrial applications, sericin peptides (3K-SP) were obtained by membrane separation and their physiochemical properties and cryoprotective function were investigated. Results showed that 3K-SP were mostly distributed less than 3000 Da, and rich in the amino acids Ser, Asp, Gly, Thr and Glu, which have been associated with the cryoprotective activity of ice-structuring proteins. Addition of 3K-SP to a frozen solution led to the reductions in melting temperature and melting time compared to control solution. In addition, 3K-SP inhibited ice recrystallization, since it could maintain small ice crystal sizes within a frozen solution. Furthermore, 3K-SP demonstrated high cryogenic protection activity of Lactobacillus delbrueckii Subsp. Bulgaricus during freezing, and provided optimal protection of cells at conditions in which its concentration was 1.0 mg/mL and the pH of the solution was 7.0. In these conditions, the percentage of surviving cells was as high as 84.78 ± 3.07%. Flow cytometric and scanning electron microscopy analyses showed that treatment with 3K-SP increased the percentage of viable cells from 52.9% to 80.1%, and suggest that 3K-SP may mediate its protective effects through interaction with cell membranes, whereby it surrounds cells in a glassy matrix that helps maintain their membrane integrity.

Optimisation of bambara groundnut water extract and skim milk composition as cryoprotectant for increasing cell viability ofLactobacillusspp. using response surface methodology

Summary Four legume water extracts, that is bambara groundnut, soya bean, red kidney bean and black bean as well as skim milk, were examined for their effectiveness in protecting Lactobacillus rhamnosus GG and Lactobacillus fermentum SK5 during the freeze-drying and storage. Bambara groundnut water extract (BGWE) showed promising cryoprotective activity that was comparable to skim milk. BGWE and skim milk at 2–10% w/v and 5–20% w/v individually produced survival rates for both strains of 87–88%. To further optimise the synergistic cryoprotective medium, response surface methodology was employed. The optimal combination was 4.93% w/v BGWE and 11.68% w/v skim milk for L. rhamnosus GG and 5.17% w/v BGWE and 11.36% w/v skim milk for L. fermentum SK5 with survival rates of 95.17% and 94.36%, respectively. The storage life of freeze-dried cells of both probiotics at 4 °C and 30 °C for 6 months was markedly improved when they were produced with these optimal combinations.

Inhibition of ice recrystallization and cryoprotective activity of wheat proteins in liver and pancreatic cells.

Efficient cryopreservation of cells at ultralow temperatures requires the use of substances that help maintain viability and metabolic functions post-thaw. We are developing new technology where plant proteins are used to substitute the commonly-used, but relatively toxic chemical dimethyl sulfoxide. Recombinant forms of four structurally diverse wheat proteins, TaIRI-2 (ice recrystallization inhibition), TaBAS1 (2-Cys peroxiredoxin), WCS120 (dehydrin), and TaENO (enolase) can efficiently cryopreserve hepatocytes and insulin-secreting INS832/13 cells. This study shows that TaIRI-2 and TaENO are internalized during the freeze-thaw process, while TaBAS1 and WCS120 remain at the extracellular level. Possible antifreeze activity of the four proteins was assessed. The "splat cooling" method for quantifying ice recrystallization inhibition activity (a property that characterizes antifreeze proteins) revealed that TaIRI-2 and TaENO are more potent than TaBAS1 and WCS120. Because of their ability to inhibit ice recrystallization, the wheat recombinant proteins TaIRI-2 and TaENO are promising candidates and could prove useful to improve cryopreservation protocols for hepatocytes and insulin-secreting cells, and possibly other cell types. TaENO does not have typical ice-binding domains, and the TargetFreeze tool did not predict an antifreeze capacity, suggesting the existence of nontypical antifreeze domains. The fact that TaBAS1 is an efficient cryoprotectant but does not show antifreeze activity indicates a different mechanism of action. The cryoprotective properties conferred by WCS120 depend on biochemical properties that remain to be determined. Overall, our results show that the proteins' efficiencies vary between cell types, and confirm that a combination of different protection mechanisms is needed to successfully cryopreserve mammalian cells.

Overexpression of Arabidopsis NADPH-dependent thioredoxin reductase C (AtNTRC) confers freezing and cold shock tolerance to plants

Overexpression of AtNTRC (AtNTRCOE) in Arabidopsis thaliana led to a freezing and cold stress tolerance, whereas a knockout mutant (atntrc) showed a stress-sensitive phenotype. Biochemical analyses showed that the recombinant AtNTRC proteins exhibited a cryoprotective activity for malate dehydrogenase and lactic dehydrogenase. Furthermore, conclusive evidence of its interaction with nucleic acids in vitro is provided here on the basis of gel shift and electron microscopy analysis. Recombinant AtNTRC efficiently protected RNA and DNA from RNase A and metal catalyzed oxidation damage, respectively. The C-terminal thioredoxin domain is required for the nucleic acid–protein complex formation. From these results, it can be hypothesized that AtNTRC, which is known to be an electron donor of peroxiredoxin, contributes the stability of macromolecules under cold stress.

Hypothermia protection effect of antifreeze peptides from pigskin collagen on freeze-dried Streptococcus thermophiles and its possible action mechanism

Antifreeze peptides (APP) obtained from pigskin collagen hydrolyzates, with molecular mass distribution among 150–2000 Da, rich in glycine (23%), proline (15%), hydroxyproline (13%) and alanine (8%), showed a high hypothermia protection activity on lactic acid bacteria during freeze-drying process. In this study, we investigated the cryoprotective activity on Streptococcus thermophiles in the presence of APP. The optimum condition for APP to protect the cells was 1 mg/mL under pH 9.0. When the S. thermophiles were dried together with 1 mg/mL of APP, 71% of organism cells survived. The intracellular proteins, nucleic acid and the key enzyme activities were determined to elucidate the possible action mechanism, and the results showed that the intracellular proteins and nucleic acid were more stable in the APP-treated group than those in the groups treated by sugars and skim milk as cryoprotectants. Moreover, the activities of lactate dehydrogenase and β-galactosidase were both higher in the APP-treated group than those in control groups. Under the investigation of SEM, cells of APP-treated group were significantly full and integral, while cells in control group were shrinking. FTIR studies implied that APP had interactions with the cell membrane phospholipids during freeze drying.

New emulsifying and cryoprotective exopolysaccharide from Antarctic Pseudomonas sp. ID1

Pseudomonas sp. ID1 is a cold-adapted bacterium isolated from a marine sediment sample collected from South Shetland Islands (Antarctica) that is noted for the highly mucous appearance of its colonies. In this work, we have characterized an exopolysaccharide (EPS) produced by this strain, which is mainly composed of glucose, galactose and fucose, and has a molecular mass higher than 2×106Da. We have also studied its potential biotechnological applications as an emulsifier and cryoprotectant agent. The EPS emulsifying activity against different food and cosmetic oils was much higher than commercial gums such as xanthan gum and arabic gum, and surfarctants such as Span 20. It formed highly stable emulsions against the cosmetic oil cetiol V, exhibiting pseudoplastic flow behavior, low thixotrophy and yield stress. The EPS of Pseudomonas sp. ID1 conferred significant cryoprotection for the strain itself as well as for other bacteria, including Escherichia coli, suggesting a universal cryoprotectant role. The cryoprotective activity of the EPS showed a clear dose–response relation at −20°C and −80°C and was significantly higher than that observed for the membrane stabilizer fetal bovine serum (FBS). These properties make the EPS of Pseudomonas sp. ID1 a promising alternative to commercial polysaccharides as an emulsifier and cryoprotectant agent for food, pharmaceutical and cosmetic industries.

Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae.

The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1, which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.

Efficient purification of cryoprotective dehydrin protein from the radish (Raphanus sativus) taproot

Dehydrin is a protein that is related to cold stress tolerance in plants. Because dehydrin shows potent cryoprotective activity, it has the potential to be used in food storage applications. In this paper, we presented an efficient purification method for native dehydrin from radishes (Raphanus sativus). Immunoblot analysis using an anti-Arabidopsis KS-type dehydrin antibody revealed that the related dehydrin accumulates in the radish taproot. The radish dehydrin that accumulated in the vascular tissues of the taproot was purified through two simple chromatographic steps: immobilized metal affinity chromatography followed by anion exchange chromatography. The yield was higher than yields previously reported on a fresh weight basis. The cryoprotective activity for malate dehydrogenase shown by purified dehydrin was more potent than that shown by small molecule cryoprotectants. This suggests that the radish is an appropriate source for the production of native dehydrin.

The influence of ultra-low temperatures on marine microalgal cells

The development of cryopreservation methods for microalgae opens great prospects for marine biotechnology and aims to establish a bank of cryopreserved cultures. Eight of ten marine microalgae species used in this study (the diatoms, green, red, and golden algae), including five previously untested species, were successfully recovered after freezing to ultra-low temperatures (−196 °C) using penetrating (dimethyl sulfoxide, glycerol, and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants. We found that ethylene glycol in combination with trehalose possessed the most effective cryoprotective activity among the algae cryoprotectants tested. However, the chief factor for the successful preservation of microalgal cells during freeze–thawing was shown to be the cooling rate. Cooling was performed in two ways: step or fast droplet freezing. The droplet freezing described here was effective only for cryopreserving green algae, whereas step freezing was optimal for all other algal species. Three diatoms of the genus Attheya were successfully cryopreserved for the first time, but none of the tested protocols had a positive result for the diatoms belonging to Pseudo-nitzschia. The failure may be explained rather by peculiarities in the cell wall composition (higher content of silica and fewer organic components) than by the specific (long and thin) shape of these cells. The pigment content in all of the studied species tended to decrease after thawing as compared with unfrozen cells and increase significantly during cell recovery. Cryosensitivity of marine algae depended on the differences in natural intrinsic characteristics rather than their taxonomic position.

Two cold-induced family 19 glycosyl hydrolases from cherimoya (Annona cherimola) fruit: An antifungal chitinase and a cold-adapted chitinase

Two cold-induced chitinases were isolated and purified from the mesocarp cherimoyas (Annona cherimola Mill.) and they were characterised as acidic endochitinases with a Mr of 24.79 and 47.77kDa (AChi24 and AChi48, respectively), both family 19 glycosyl hydrolases. These purified chitinases differed significantly in their biochemical and biophysical properties. While both enzymes had similar optimal acidic pH values, AChi24 was enzymatically active and stable at alkaline pH values, as well as displaying an optimal temperature of 45°C and moderate thermostability. Kinetic studies revealed a great catalytic efficiency of AChi24 for oligomeric and polymeric substrates. Conversely, AChi48 hydrolysis showed positive co-operativity that was associated to a mixture of different functional oligomeric states through weak transient protein interactions. The rise in the AChi48 kcat at increasing enzyme concentrations provided evidence of its oligomerisation. AChi48 chitinase was active and stable in a broad acidic pH range, and while it was relatively labile as temperatures increased, with an optimal temperature of 35°C, it retained about 50% of its maximal activity from 5 to 50°C. Thermodynamic characterisation reflected the high kcat of AChi48 and the remarkably lower ΔH(‡), ΔS(‡) and ΔG(‡) values at 5°C compared to AChi24, indicating that the hydrolytic activity of AChi48 was less thermodependent. In vitro functional studies revealed that AChi24 had a strong antifungal defence potential against Botrytis cinerea, whereas they displayed no cryoprotective or antifreeze activity. Hence, based on biochemical, thermodynamic and functional data, this study demonstrates that two acidic endochitinases are induced at low temperatures in a subtropical fruit, and that one of them acts in an oligomeric cold-adapted manner.

Laboratory-scale extraction and characterization of ice-binding sericin peptides

Sericin peptides (SPs) are a new cryoprotectant with protecting abilities for organisms or biomacromolecule in food stuff from freezing injury in subzero environments. The extraction of ice-binding sericin peptides (I-SPs) by ice affinity extraction adsorption and their cryoprotective effect on Lactobacillus Delbrueckii Subsp. bulgaricus LB340 LYO during frozen storage was studied in this paper. The optimal extraction conditions by application of a new and assembled ice absorption apparatus were identified as temperature of −3 °C, SPs concentration of 5 mg/mL, and extraction time of 3 h. Compared with SPs, I-SPs were found to be increased in the contents of some special amino acid residues, including Ser, Thr, Gly, and Ala, which might be relative with the ice affinity as well as the cryoprotective activity of ice-structuring proteins. Meanwhile, the molecular mass distribution data implied that the free amino acids in the SPs could be excluded from the ice affinity adsorption, and peptides containing 3–6 amino acid residues might be more liable to adsorb to ice surface. Furthermore, with addition of I-SPs at concentrations of more than 0.01 mg/mL, LB340 strain had a higher survival rate after frozen storage compared to the water control as well as SPs (p < 0.05).

Dehydrins (LTI29, LTI30, COR47) from Arabidopsis thaliana expressed in Escherichia coli protect Thylakoid membrane during freezing

As the name dehydrins implies, these proteins are typically expressed in response to dehydration which can be caused by drought, osmotic stress or freezing temperatures 1. In general, dehydrins occur in plants as multi-gene families. Four Arabidopsis dehydrins (LTI29, ERD14, COR47 and RAB18) have been tested for protection of thylakoid membranes during freeze thaw cycle in vitro. 2,3 Our firstly reported results show that dehydrins LTI29, ERD14, COR47 have cryoprotective activity while RAB18 did not protect the thylakoid membranes at low temperatures. The cryoprotective activity reached a maximum of 50% to 60% at protein concentration of 140-250 ?g/ml in the assay. A contribution of dehydrins to freezing tolerance in vivo is supported by the observation of Nylander et al. (2001)4 that LTI29 and COR47 are cold induced at mRNA and protein expression level.

Leaf seasonal accumulation of a 47-kDa dehydrin and changes in its cryoprotective activity in Nothofagus dombeyi (Mirb.) Blume

Nothofagus dombeyi (Mirb.) Blume is an evergreen tree, pioneer in habitats subject to unfavorable environmental conditionsas nighttime freezing temperatures and low water content. It inhabits zones characterized by cold Winter with freezingtemperature and Spring frosts reaching to -5oC. Furthermore, it has the capacity to maintain a functional photosyntheticapparatus at low temperatures. We postulated that N. dombeyi is capable to accumulate dehydrins in leaves in response tocold. Seasonal variation in dehydrins, soluble total proteins, and cryoprotective activity in vitro of protein extracts of N.dombeyi leaves were analyzed during the years 2004 and 2005. A dehydrin-like protein of 47-kDa was immunologicallyidentified; this protein was present only in Winter and Spring. The highest accumulation of this protein and the maximalin vitro cryoprotective activity was found during Spring. The leaf total protein extract of this season preserved the 100%of lactate dehydrogenase (LDH) enzyme activity after 5 freeze/thaw cycles in liquid nitrogen adding 10 ?g/ml of totalproteins extract. The soluble protein concentration increased from 1.8 mg/g in Summer to 4.8 mg/g in Fall on base offresh weight, and declined through Winter and Spring. The 47-kDa dehydrin may be related to cold hardiness of leaftissue in overwintering N. dombeyi. We conclude that cell dehydration caused by freezing is the main factor involved inthe triggering of 47-kDa dehydrin accumulation in leaves of N. dombeyi. The accumulation of reactive oxygen species(ROS) by oxidative stress product of high irradiance and frosts during Spring, and their role in the triggering of the 47-kDadehydrin accumulation in leaves of N. dombeyi is discussed.

Traditional and Modern Protective Media for the Low-Temperature Bacteria Preservation

Presented are the literature data on the efficiency of bacteria preservation at temperatures ranging from -20 to -196 °C in the protective media containing such cryoprotectors as glycerol, dimethyl sulfoxide, carbo-hydrates, substances of protein origin specified by regulatory guidelines. Most of the focus is on the publications reporting the results of the long-term bacteria preservation at sub-zero temperatures, optimization of the protective media for pathogenic bacteria, and usage of the compounds with potential cryoprotective activity. Noted is the necessity for approbation of the specified protective media for the conserved bacteria species at the applied preserving temperatures. One of the approaches to the enhancement of the low-temperature preservation techniques is a search for natural protectors, which can provide for surviving of bacteria in the unfavorable conditions, including low temperatures, and a search for possibility to integrate these natural protectors into the cryoprotective media. Displayed are the results of effective application of glycerol-betaine, and polysaccharides of Arctic microorganisms for the low-temperature bacteria preservation.

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya ( Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO 2-treated cherimoya fruit ( Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a M r of 22.07 kDa and a p I of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k cat (3.10 ± 0.04 s −1) and K m (0.32 ± 0.03 mg ml −1) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k cat at both 37 and 5 °C, and a low E a (6.99 kJ mol −1) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze–thaw inactivation of lactate dehydrogenase. Indeed, the PD 50 was 8.7 μg ml −1 (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO 2-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO 2.

Factors influencing cryoprotective activity and drug leakage from liposomes after freezing

The effects of freezing and thawing conditions and cryoprotective additives on release of streptomycin from lecithin liposomes following freeze-thaw cycles have been investigated. Drug retention was maximized by slow cooling (approx. 1 degree C min-1). At temperatures between 0 degree and -20 degrees C, the extent of drug loss was time-dependent indicating incomplete freezing; below -45 degrees C this effect was abolished and the system was stable. Osmotic gradients across the liposome membrane during freezing were found to have little effect on drug loss. Marked cryoprotective activity was shown by dimethylsulphoxide, glycerol, alanine and glycinebetaine at concentrations of 3% w/v or less. At this concentration sucrose and mannitol had little activity.

Potent cryoprotective activity of cold and CO 2-regulated cherimoya ( Annona cherimola) endochitinase

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO 2-treated cherimoya fruit ( Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a M r of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 °C, and it retained about 30% of its maximum activity at 5 °C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 °C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high k cat (6.93 s −1) value for the fluorogenic substrate 4-MU-(GlcNAc) 3, reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k cat at 37 and 5 °C, and a low E a (11.32 kJ mol −1). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze–thaw inactivation of lactate dehydrogenase. The PD 50 was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO 2-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO 2 concentrations.

The fatty acid profile changes in marine invertebrate larval cells during cryopreservation

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to −196°C using traditional cryoprotectants (Me2SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me2SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (α-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas–liquid chromatography. The results indicate that freezing–thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing–thawing.

Comparative expression and transcript initiation of three peach dehydrin genes

Dehydrin genes encode proteins with demonstrated cryoprotective and antifreeze activity, and they respond to a variety of abiotic stress conditions that have dehydration as a common component. Two dehydrins from peach (Prunus persica L. [Batsch.]) have been previously characterized; here, we describe the characterization of a third dehydrin from peach bark, PpDhn3, isolated by its response to low temperature. The expression of all three dehydrin genes was profiled by semi-quantitative reverse transcription PCR, and transcript initiation was mapped for all three genes using the RNA ligase-mediated 5' rapid amplification of cDNA ends technique. PpDhn3 transcripts from bark collected in December or July, as well as transcripts from developing fruit, initiated at a single site. Although most of the PpDhn1 transcripts initiated at a similar position, those from young fruit initiated much further upstream of the consensus TATA box. Bark and fruit transcripts encoding PpDhn2 initiated ca. 30 bases downstream of a consensus TATA box; however, transcripts from ripe fruit initiated further upstream. Ripe fruit transcripts of PpDhn2 contain a 5' leader intron which is predicted to add some 34 amino acids to the N-terminal methionine of the cognate protein when properly processed. Secondary structure prediction of sequences surrounding the TATA box suggests that conformational transitions associated with decreasing temperature contribute to the regulation of expression of the cold-responsive dehydrin genes. Taken together these results reveal new, unexpected levels of gene regulation contributing to the overall expression pattern of peach dehydrins.

Evaluation of the Protective Activities of a Late Embryogenesis Abundant (LEA) Related Protein, Cor15am, during Various Stressesin Vitro

Arabidopsis Cor15am is a late embryogenesis abundant (LEA) related protein that has been shown to exhibit cryoprotective activity in vitro. In this study, we further investigated the mechanisms by which Cor15am protects substrates from inactivation. Although Cor15am did not exhibit refolding activity, it showed protective activity against various stresses in vitro. This might be attributable to the activity of Cor15am in attenuating the aggregation of the substrates. Our data indicate that Cor15am functions as a protectant against various stresses by preventing protein aggregation.