Intracellular Trehalose Concentration Research Articles

The Relationship Between Tolerance and Respond of Saccharomyces cerevisiae Strains Against Different Stress Treatments

This study evaluated the stress tolerance and stress responds of three indigenous Saccharomyces cerevisiae strains and one reference strain against temperature (45°C, 50°C, and 55°C) and ethanol (12 and 14%) stresses over time by assessing the yeast counts and intracellular trehalose content. The maximum decrease in the yeast counts was at 55°C and 14%, and also, 45°C and 14% caused highest intracellular trehalose content for all strains when each treatment evalulated among its variables (P < 0.05). The correlation between the trehalose content and Log N levels of the strains was significant with the correlation coefficient (r) 0.62 and 0.39 for strains 2 and 7 against temperature stress, also a strong and significant correlation was determined with r values for strains 2,7, 47 against ethanol stress, respectively (P < 0.05).

Effects and molecular mechanism of sugar transporter ESA_RS15745 on desiccation resistance, motility, and biofilm formation of Cronobacter sakazakii.

Cronobacter sakazakii, an important Gram-negative foodborne pathogen, can cause neonatal meningitis and sepsis with high rates of infection and death. Gene ESA_RS15745 encodes a sugar transporter protein, which is not only essential for osmotic pressure maintenance during bacterial growth and reproduction but also associated with their desiccation tolerance, motility, and biofilm formation. Here, a mutant strain of ESA_RS15745 (ΔESA_RS15745) and the complementation strain (cpESA_RS15745) were constructed using a suicide vector knockout and gene complementation. ΔESA_RS15745 was found to have a decrease in its ability to transport maltose and trehalose and resist desiccation, whereas an increase in the ability of motility and biofilm formation, implying that ESA_RS15745 may positively regulate sugar transport and desiccation tolerance and negatively regulate motility and biofilm formation. To further investigate the molecular mechanisms underlying the function of related genes, RNA-seq was performed to explore the differentially expressed genes in the mutants. RNA-seq results showed the upregulation of 114 genes (mainly including those regulating chemotaxis and flagellar motility) and the downregulation of 22 genes (mainly including those regulating sugar transport). qRT-PCR analysis supported the RNA-seq results and showed that ESA_RS15745 may influence the dehydration tolerance though decreasing the intracellular trehalose content and negatively regulate the motility though the chemotactic signaling pathway. In addition, the biofilm formation of C. sakazakii should also be speculated to negatively regulate by ESA_RS15745 by consuming the extracellular carbohydrates concentration and then downregulating the intracellular cyclic diguanosine monophosphate. This study offers a reference for comprehending the molecular mechanism of gene ESA_RS15745 in C. sakazakii.

Genome-Wide Scanning Loci and Differentially Expressed Gene Analysis Unveils the Molecular Mechanism of Chlorantraniliprole Resistance in Spodoptera frugiperda.

Chlorantraniliprole has been widely used to controlSpodoptera frugiperda, but it has led to the development of chlorantraniliprole resistance. Multiomics analysis of strains with two extreme traits helps to elucidate the complex mechanisms involved. Herein, following genome resequencing and application of the Euclidean distance algorithm, 550 genes within a 16.20-Mb-linked region were identified from chlorantraniliprole-resistant (Ch-R) and chlorantraniliprole-susceptible (Ch-Sus) strains. Using transcriptome sequencing, 2066 differentially expressed genes were identified between Ch-R and Ch-Sus strains. Through association analysis, three glutathione S-transferase family genes and four trehalose transporter genes were selected for functional verification. Notably, SfGSTD1 had the strongest binding ability with chlorantraniliprole and is responsible for chlorantraniliprole tolerance. The Ch-R strain also increased the intracellular trehalose content by upregulating the transcription of SfTret1, thereby contributing to chlorantraniliprole resistance. These findings provide a new perspective to reveal the mechanism of resistance of agricultural pests to insecticides.

Intracellular trehalose accumulation via the Agt1 transporter promotes freeze-thaw tolerance in Saccharomyces cerevisiae.

This study is to investigate the use of a constitutively expressed trehalose transport protein to directly control intracellular trehalose levels and protect baker's yeast (Saccharomyces cerevisiae) cells against freeze-thaw stress in vivo. We used a constitutively overexpressed Agt1 transporter to investigate the role of trehalose in the freeze-thaw tolerance of yeast cells by regulating intracellular trehalose concentrations independently of intracellular biosynthesis. Using this method, we found that increasing intracellular trehalose in yeast cells improved cell survival rate after 8 days of freezing at -80 and -20°C. We also observed that freeze-thaw tolerance promoted by intracellular trehalose only occurs in highly concentrated cell pellets rather than cells in liquid suspension. Trehalose is sufficient to provide freeze-thaw tolerance using our Agt1 overexpression system. Freeze-thaw tolerance can be further enhanced by deletion of genes encoding intracellular trehalose degradation enzymes. These findings are relevant to improving the freeze-thaw tolerance of baker's yeast in the frozen baked goods industry through engineering strains that can accumulate intracellular trehalose via a constitutively expressed trehalose transporter and inclusion of trehalose into the growth medium.

Screening and transcriptomic analysis of the ethanol-tolerant mutant Saccharomyces cerevisiae YN81 for high-gravity brewing.

Ethanol stress is one of the major limiting factors for high-gravity brewing. Breeding of yeast strain with high ethanol tolerance, and revealing the ethanol tolerance mechanism of Saccharomyces cerevisiae is of great significance to the production of high-gravity beer. In this study, the mutant YN81 was obtained by ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis from parental strain CS31 used in high-gravity craft beer brewing. The ethanol tolerance experiment results showed that cell growth and viability of YN81 were significantly greater than that of CS31 under ethanol stress. The ethanol tolerance mechanisms of YN81 were studied through observation of cell morphology, intracellular trehalose content, and transcriptomic analysis. Results from scanning electron microscope (SEM) showed alcohol toxicity caused significant changes in the cell morphology of CS31, while the cell morphology of YN81 changed slightly, indicating the cell morphology of CS31 got worse (the formation of hole and cell wrinkle). In addition, compared with ethanol-free stress, the trehalose content of YN81 and CS31 increased dramatically under ethanol stress, but there was no significant difference between YN81 and CS31, whether with or without ethanol stress. GO functional annotation analysis showed that under alcohol stress, the number of membrane-associated genes in YN81 was higher than that without alcohol stress, as well as CS31, while membrane-associated genes in YN81 were expressed more than CS31 under alcohol stress. KEGG functional enrichment analysis showed unsaturated fatty acid synthesis pathways and amino acid metabolic pathways were involved in ethanol tolerance of YN81. The mutant YN81 and its ethanol tolerance mechanism provide an optimal strain and theoretical basis for high-gravity craft beer brewing.

Proteomics Analysis of Zygosaccharomyces mellis in Response to Sugar Stress

The high-osmotic-pressure environment of honey is not suitable for the survival of microorganisms, except for osmotic-tolerant fungal and bacterial spores. In this study, shotgun metagenomic sequencing technology was used to identify yeast species present in honey samples. As a result, Zygosaccharomyces spp. yeast, including Zygosaccharomyces rouxii, Z. mellis and Z. siamensis, were isolated. The intracellular trehalose and glycerin concentrations of yeast, as well as the antioxidant-related CAT, SOD and POD enzyme activities, increased under a high-glucose environment (60%, w/v). To learn more about the osmotic resistance of Z. mellis, iTRAQ-based proteomic technology was used to investigate the related molecular mechanism at the protein level, yielding 522 differentially expressed proteins, of which 303 (58.05%) were upregulated and 219 (41.95%) were downregulated. The iTRAQ data showed that the proteins involved in the pathway of the cell membrane and cell-wall synthesis, as well as those related to trehalose and glycerin degradation, were all downregulated, while the proteins in the respiratory chain and TCA cycle were upregulated. In addition, formate dehydrogenase 1 (FDH1), which is involved in NADH generation, displayed a great difference in response to a high-sugar environment. Furthermore, the engineered Saccharomyces cerevisiae strains BY4741△scFDH1 with a knocked-out FDH1 gene were constructed using the CRISPR/Cas9 method. In addition, the FDH1 from Z. mellis was expressed in BY4741△scFDH1 to construct the mutant strain BY4717zmFDH1. The CAT, SOD and POD enzyme activities, as well as the content of trehalose, glycerin, ATP and NADH, were decreased in BY4741△scFDH1. However, those were all increased in BY4717zmFDH1. This study revealed that Z. mellis could increase the contents of trehalose and glycerin and promote energy metabolism to improve hypertonic tolerance. In addition, FDH1 had a significant effect on yeast hypertonic tolerance.

Effect of nitrogen source on mycelial growth, blastospore production and thermotolerance of Cordyceps fumosorosea (Hypocreales: Cordycipitaceae)

Among the entomogenous fungi, Cordyceps fumosorosea has great potential for the control of piercing-sucking insects in greenhouses. The blastospores of C. fumosorosea developed in liquid fermentation are more efficient for industrialization because they can kill insects faster and be produced faster than conidia from solid cultivation. Thermotolerance of blastospores plays a major role in their application in greenhouses. The added nutrient elements during its cultivation process have a significant impact on the thermotolerance of blastospores. Understanding the effects of nitrogen sources on the mycelial growth, sporulation and thermotolerance of C. fumosorosea is essential for its commercial application. Frequently used nitrogen sources were investigated in fermentation medium, including peptone, soya peptone, yeast extract, ammonium sulfate, urea and so on. Soya peptone provided the maximum blastospore yield and GT50, which were 5.17 × 108 blastospores mL−1 and 1.91 h, respectively. To explore the difference in intracellular substances involved in the thermotolerance of blastospores in the presence of different nitrogen sources, intracellular trehalose and mannitol were extracted and then qualified by ion chromatography. The results showed that blastospores with better thermotolerance generally had lower intracellular trehalose content, indicating that the intracellular trehalose content of blastospores could be a factor that the fungus responds to thermal stress and that its content in the blastospores could be adjusted by the supplemented nitrogen source.

Trehalose accumulation and radiation resistance due to prior heat stress in Saccharomyces cerevisiae

In this study, we examined the accumulation of trehalose, a stress-responsive substance, upon gamma-ray irradiation by evaluating the cause of trehalose accumulation and the development of gamma-ray resistance through intracellular trehalose accumulation. Saccharomyces cerevisiae cells cultured to the logarithmic growth phase were irradiated with gamma rays, and the intracellular trehalose content was measured. However, trehalose was not detectable. The yeast cells with trehalose accumulation caused by pre-treatment at 40°C were irradiated with gamma rays, and the resistance of these cells to gamma radiation was compared with that of cells without heat treatment. Trehalose accumulation resulted in gamma-ray resistance and suppressed the increase in reactive oxygen species, lipid peroxidation, and DNA double-strand break production in yeast cells. The tests were also performed with a trehalose-6-phosphate-synthase (TPS1)-deficient mutant strain (Δtps1) unable to synthesize trehalose, and the results revealed that TPS1 was involved in protection against oxidative stress.

Oligosaccharide-phenolic compound conjugates in soluble polysaccharides from rice straw alleviate ethanol fermentation stresses in Saccharomyces cerevisiae

In this study, it was found that soluble polysaccharides (SPs) from rice straw were conjugated with high levels of phenolic compounds. A gradient ethanol precipitation method was used to separate conjugates with different phenolic compound components. These conjugates were mainly composed of disaccharides or trisaccharides and phenolic acids that were mainly p-coumaric acid, ferulic acid, vanillic acid, and vanillin. SPs with more conjugates exhibited better effects on Saccharomyces cerevisiae and ethanol fermentation. The change in the proportion of conjugates in SPs significantly affected the accumulation of intracellular trehalose and glycerol content, indicating the effect of the conjugates on intracellular osmotic stress. Moreover, SPs with more conjugates generated lower intracellular hydrogen peroxide (H2O2) levels. Thus, oligosaccharide and phenolic compound conjugates in SPs promoted yeast growth and ethanol fermentation. For instance, OD value reached 9.4, which was 1.3 times higher than the control group, and ethanol content at 36 h was 36.5 g/L, which was 1.3 times higher than the control. Osmotic pressure alleviation and lowered intracellular H2O2 levels by conjugates are key mechanisms of the SPs during yeast growth and ethanol fermentation.

Facilitating trehalose entry into hRBCs at 4 °C by alkylated ε-poly(L-lysine) for glycerol-free cryopreservation.

Currently, glycerol is a conventional cryoprotectant of human red blood cells (hRBCs), but the time-consuming thawing and deglycerolization processes are essential before transfusion. Much of the research up to now has been conducted on the delivery of impermeable trehalose to hRBCs at 37 °C, but the cryoprotective effect of trehalose and deterioration of cells still remain challenging. Encouraged by the interaction of hydrophobic or cationic groups on cell membranes and osmotic stabilization, herein, we propose a novel cryopreservation system to facilitate trehalose entry into hRBCs at 4 °C and pH 7.4. High intracellular trehalose contents and cryosurvival of hRBCs were achieved with small function variations via the assistance of self-assembled nanoparticles of alkylated ε-poly(L-lysine) (ε-PL) along with poly(vinyl pyrrolidone) (PVP). The effect of amphipathic alkylated ε-PL with various alkyl chains and grafting ratios on membrane perturbation with protection of PVP was systematically investigated. Overall, by the combination of alkylated ε-PL and PVP, the intracellular trehalose could be enhanced to 109.7 ± 6.1 mM and subsequently hRBC cryosurvival reached 91.7 ± 5.5%, significantly higher than those containing trehalose only, 11.9 ± 1.1 mM and 50.0 ± 2.1%, respectively. It was observed that the biocompatible trehalose-loading system could benefit glycerol-free cryopreservation of hRBCs and also provide a feasible way for impermeable biomacromolecule delivery.

Influence of arginine on the biocontrol efficiency of Metschnikowia citriensis against Geotrichum citri-aurantii causing sour rot of postharvest citrus fruit

This study investigated the effect of arginine (Arg) on the antagonistic activity of Metschnikowia citriensis against sour rot caused by Geotrichum citri-aurantii in postharvest citrus, and evaluated the possible mechanism therein. Arg treatment up-regulated the PUL genes expression, and significantly induced the pulcherriminic acid (PA) production of M. citriensis, which related to the capability of iron depletion of M. citriensis. By comparing the biocontrol effects of Arg-treated and untreated yeast cells, it was found that Arg treatment significantly enhanced the biocontrol efficacy of M. citriensis, and 5 mmol L−1 Arg exerted the best effect. Additionally, the biofilm formation ability of M. citriensis was greatly enhanced by Arg, and the higher population density of yeast cells in citrus wounds was also observed in Arg treatment groups stored both at 25 °C and 4 °C. Moreover, Arg was shown to function as a cell protectant to elevate antioxidant enzyme activity [including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX)] and intracellular trehalose content to resist oxidative stress damage, that directly helped to enhance colonization ability of yeasts in fruit wounds. These results suggest the application of Arg is a useful approach to improve the biocontrol performance of M. citriensis.

Sonoporation enables high-throughput loading of trehalose into red blood cells

Despite recent advances in biostabilization, clinical blood supplies still experience shortages and storage limitations for red blood cells (RBCs) have not yet been sufficiently addressed. Storing RBCs in a frozen or dried state is an appealing solution to address storage limitations, but many promising cryoprotectants, including the non-reducing sugar trehalose, are impermeant to mammalian cell membranes and cannot be utilized effectively using currently available compound-loading methods. We found that transient pore formation induced by ultrasound and microbubbles (sonoporation) offers an effective means of loading trehalose into RBCs to facilitate long-term storage in a frozen or desiccated state. The protective potential of trehalose loading was demonstrated by freezing processed RBCs at −1 °C/min to −80 °C, then either storing the cells at −80 °C or lyophilizing them. RBCs were either thawed or rehydrated after 42 days of storage and evaluated for membrane integrity and esterase activity to estimate recovery and cell viability. The intracellular concentration of trehalose reached 40 mM after sonoporation and over 95% of treated RBCs were recovered after loading. Loading of trehalose was sufficient to maintain RBC morphology and esterase activity in most cells during freezing (>90% RBC recovery) and to a lower degree after lyophilization and rehydration (>20% recovery). Combining sonoporation with an integrated fluidics device allowed for rapid loading of up to 70 mM trehalose into RBCs. These results demonstrate the potential of sonoporation-mediated trehalose loading to increase recovery of viable RBCs, which could lead to effective methods for long-term stabilization of RBCs.

Pulsed electric field pre-treatment for enhanced bacterial survival after drying: Effect of carrier matrix and strain variability

Previous studies suggest that increased intracellular trehalose concentrations can result in increased robustness of probiotics and starter cultures after processing. We evaluated whether pulsed electric field (PEF) pre-treatment, resulting in increased intracellular trehalose concentrations, can lead to enhanced survival during spray drying and subsequent storage and what the effect of the carrier matrix during drying herein is on survival. The applied PEF pre-treatment resulted in increased survival of Lactobacillus plantarum WCFS1 after spray drying and subsequent storage in reconstituted skim milk (RSM), though not in the other evaluated carrier matrices. The same PEF pre-treatment in lactose instead of trehalose electroporation medium resulted also in an increased survival after spray drying in RSM. Further analysis of intracellular trehalose and lactose concentrations revealed that survival after spray drying cannot only be explained by intracellular trehalose and/or lactose concentrations. Experiments with other bacterial strains indicated that strain variability cannot be neglected when designing a process aimed at enhanced bacterial survival after processing. Overall, PEF pre-treatment is promising for enhancing survival of bacteria after drying and storage, though further understanding of the applicability is required for industrial application. Industrial relevance textProbiotics and starter cultures are very common in food industry. Survival of these cultures during drying processes is essential for their application. A pulsed electric field pre-treatment resulting in increased bacterial survival after spray drying and powder storage may contribute to more energy efficient production processes of dried bacterial cultures.

Temperature and presence of ethanol affect accumulation of intracellular trehalose in Lactobacillus plantarum WCFS1 upon pulsed electric field treatment

Pulsed electric field (PEF) treatment can be used to increase intracellular small molecule concentrations in bacteria, which can lead to enhanced robustness of these cells during further processing. In this study we investigated the effects of the PEF treatment temperature and the presence of 8% (v/v) ethanol in the PEF medium on cell survival, membrane fluidity and intracellular trehalose concentrations of Lactobacillus plantarum WCFS1. A moderate PEF treatment temperature of 21 °C resulted in a high cell survival combined with higher intracellular trehalose concentrations compared to a treatment at 10 and 35 °C. Interestingly, highest intracellular trehalose concentrations were observed upon supplementing the PEF medium with 8% ethanol, which resulted in more than a doubling in intracellular trehalose concentrations, while culture survival was retained. Overall, this study shows that treatment temperature and PEF medium optimization are important directions for improving molecule uptake upon PEF processing.

Comb-like Pseudopeptides Enable Very Rapid and Efficient Intracellular Trehalose Delivery for Enhanced Cryopreservation of Erythrocytes.

Cell cryopreservation plays a key role in the development of reproducible and cost-effective cell-based therapies. Trehalose accumulated in freezing- and desiccation-tolerant organisms in nature has been sought as an attractive nontoxic cryoprotectant. Herein, we report a coincubation method for very rapid and efficient delivery of membrane-impermeable trehalose into ovine erythrocytes through reversible membrane permeabilization using pH-responsive, comb-like pseudopeptides. The pseudopeptidic polymers containing relatively long alkyl side chains were synthesized to mimic membrane-anchoring fusogenic proteins. The intracellular trehalose delivery efficiency was optimized by manipulating the side chain length, degree of substitution, and concentration of the pseudopeptides with different hydrophobic alkyl side chains, the pH, temperature, and time of incubation, as well as the polymer-to-cell ratio and the concentration of extracellular trehalose. Treatment of erythrocytes with the comb-like pseudopeptides for only 15 min yielded an intracellular trehalose concentration of 177.9 ± 8.6 mM, which resulted in 90.3 ± 0.7% survival after freeze-thaw. The very rapid and efficient delivery was found to be attributed to the reversible, pronounced membrane curvature change as a result of strong membrane insertion of the comb-like pseudopeptides. The pseudopeptides can enable efficient intracellular delivery of not only trehalose for improved cell cryopreservation but also other membrane-impermeable cargos.

Regulation of trehalose, a typical stress protectant, on central metabolisms, cell growth and division of Saccharomyces cerevisiae CEN.PK113-7D

Trehalose could protect the typical food microorganism Saccharomyces cerevisiae cell against environmental stresses; however, the other regulation effects of trehalose on yeast cells during the fermentation are still poorly understood. In this manuscript, different concentrations (i.e., 0, 2 and 5% g/v) of trehalose were respectively added into the medium to evaluate the effect of trehalose on growth, central metabolisms and division of S. cerevisiae CEN.PK113-7D strain that could uptake exogenous trehalose. Results indicated that addition of trehalose could inhibit yeast cell growth in the presence or absence of 8% v/v ethanol stress. Exogenous trehalose inhibited the glucose transporting efficiency and reduced intracellular glucose content. Simultaneously, increased intracellular trehalose content destroyed the steady state of trehalose cycle and caused the imbalance between the upper glycolysis part and the lower part, thereby leading to the dysfunction of glycolysis and further inhibiting the normal yeast cell growth. Moreover, energy metabolisms were impaired and the ATP production was reduced by addition of trehalose. Finally, exogenous trehalose-associated inhibition on yeast cell growth and metabolisms delayed cell cycle. These results also highlighted our knowledge about relationship between trehalose and growth, metabolisms and division of S. cerevisiae cells during fermentation.

Enhancing freeze resilience of baker's yeast and designing its product formulation

Trehalose loading and cold acclimatization strategies were utilized to enhance the freeze resilience of baker's yeast (Saccharomyces cerevisiae) isolate and production protocol for formulation was planned. Loading with 1.0M trehalose following 7 days of incubation prompted increase in intracellular trehalose content from 0.3% to 4.71%. Comparative freeze resistance investigations uncovered that 14 day cold acclimatized culture displays extreme cell viability (193cfu/ml) under refrigerated conditions. The 28 days acclimatized culture indicated 36.29% sugar content followed by 1.0M trehalose loaded culture (29.44%). In 1.0M trehalose loaded culture, greatest trehalose content (4.71%) and least invertase activity (29.79 nmol/min/ml) was observed. Biomass production of baker's yeast was done at conditions pre-optimized in our laboratory: pH-6.0, aeration-120 rpm and temperature-30⁰C utilizing unclarified molasses as carbon source and corn steep liquor (CSL) as a nitrogen source. Best dough rising capacity was shown by 14 days cold acclimatized strain. Product formulation as cream yeast and dried (lyophilized and plate dried) yeast was done, out of which cream yeast was found appropriate for bread making while nutritional profiling of dried yeast formulations revealed it as a good source of protein, minerals and essential amino acids.

Enhancing spray drying tolerance of Lactobacillus bulgaricus by intracellular trehalose delivery via electroporation.

Spray-drying is an efficient drying technique for preparing probiotic powders, but the high temperatures employed during the process results in low survival rates of lactic acid bacteria. This study aimed to enhance spray-drying tolerance of lactic acid bacteria by intracellular delivery of trehalose via electroporation. Diverse electroporation conditions were applied to Lactobacillus delbrueckii ssp. bulgaricus sp1.1 cells exposed to 10% trehalose prior to spray drying with 30% re-constituted skimmed milk. Survival rates of spray-dried bacteria increased with an intracellular trehalose content of ≥3.5 µg/107 colony-forming units (CFU) and reached 100% with intracellular trehalose content of 10.1 µg/107 CFU. The application of two electroporation pulses at 2.5 kV/cm helped increase the survival rate of L. bulgaricus from 38% to 61% after spray drying. Membrane damage and pore formation caused by excessive pulsed electric field treatment resulted in cell death after spray drying. Sufficient intracellular trehalose protected cell walls and membranes from further damage by spray drying. Multi-pulse electroporation of trehalose into lactic acid bacteria prior to spray drying can potentially increase cell viability.

Trehalose uptake and dehydration effects on the cryoprotection of CHO–K1 cells expressing TRET1

Chinese hamster ovary cells (CHO–K1 cells) in which the trehalose transporter (TRET1) is expressed can have greater cryoprotection than ordinary CHO–K1 cells. This study examines the uptake characteristics of trehalose into cells via TRET1 and determines the influence of intracellular trehalose on the freeze–thaw viabilities. In our experiments, the intracellular trehalose concentration is controlled by the extracellular trehalose concentration and the immersion time in a freezing solution. In this freezing solution, both kinds of CHO–K1 cells are independently dispersed with various amount of trehalose, and then put into the CO2 incubator for 0–6 h. After a set immersion time, the cell-suspended sample is cooled to 193 K, stored for 1 week, then quickly thawed at 310 K and its viability measured. The uptake amount of intracellular trehalose is measured before freezing. We find an upper limit for the uptake amount of trehalose when the extracellular trehalose concentration is about 400 mM, at which the freeze–thaw viability is the highest. When the extracellular trehalose concentration exceeds 400 mM, shorter immersion times are needed to obtain the maximum freeze–thaw viability. Also, longer immersion weakens the cells. Our analyses indicate that when the extracellular trehalose-concentration is less than 400 mM, the trehalose uptake occurs more slowly with less dehydration, resulting in less stress on the cell. When the extracellular trehalose concentration exceeds the saturation level, the cell is stressed by the excess dehydration due to the remaining osmotic pressure, with apoptosis occurring before freezing.

Accumulation of intracellular trehalose and lactose in Lactobacillus plantarum WCFS1 during pulsed electric field treatment and subsequent freeze and spray drying

Survival of bacteria during drying processes is required for products such as probiotics or starter cultures. In earlier studies high intracellular trehalose concentrations have been related to processing robustness during freezing and drying for several cell types. In this study we evaluated pulsed electric field (PEF) pre-treatment to increase intracellular trehalose in Lactobacillus plantarum WCFS1 and subsequent survival during freeze drying and storage in a trehalose matrix. Surprisingly, despite increased intracellular trehalose by PEF pre-treatment no enhanced survival was observed after drying and storage. Additional analysis of intracellular trehalose before and after freeze drying and spray drying revealed that during the drying process intracellular trehalose concentrations increased. Due to this increase the intracellular trehalose concentrations in the PEF pre-treated and control samples did not differ anymore. To explain what happens during the drying processes, results for trehalose were compared to similar experiments with lactose. A remarkable difference was observed between transfer of trehalose and lactose into cells during the freezing step before freeze drying, while both disaccharides gave similar cellular protection when used as freezing and drying matrix. Generated knowledge on transport of disaccharides into bacterial cells during freezing and drying can benefit processing of living bacterial formulations.