Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs.

Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts. During storage of frozen doughs, strains of T. delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days. Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain. Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323. These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions. The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S. cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T. delbrueckii (IGC 5321 and IGC 5323). However, the strains of S. cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.     

Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage

Thermophilic lactic acid bacteria exhibit different survival rates during freezing and frozen storage, depending on the processing conditions. We used a Plackett and Burman experimental design to study the effects of 13 experimental factors, at two levels, on the resistance of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to freezing and frozen storage. The resistance was evaluated by quantifying the decrease of acidification activity during freezing and throughout 8 weeks of storage. Acidification activity after freezing and frozen storage was affected by 12 experimental factors. Only the thawing temperature did not show any significant effect. S. thermophilus was more resistant than L. bulgaricus and the cryoprotective effect of glycerol during freezing and storage was confirmed. The temperature and duration of the cryoprotection step influenced acidification activity following the freezing step: the lower the temperature and the shorter the duration, the higher the activity. Acidification activity after storage was affected by several experimental factors involved in the fermentation stage: use of NaOH instead of NH4OH for pH control, addition of Tween 80 in the culture medium, and faster cooling led to better cryotolerance. Resistance to freezing and frozen storage was improved by using a high freezing rate and a low storage temperature. Finally, this study revealed that the conditions under which lactic acid bacteria are prepared should be well controlled to improve their preservation and to limit the variability between batches and between species.     

Microfiltration conditions modify Lactobacillus bulgaricus cryotolerance in response to physiological changes

This work aimed at analyzing the effect of microfiltration conditions (cross-flow velocity and transmembrane pressure) on the quality of frozen Lactobacillus bulgaricus CFL1 starters produced on pilot scale. Microfiltered cells were less resistant during the concentration process than centrifuged cells. In contrast, bacterial cryotolerance during freezing was improved after microfiltration, in a range of 28–88%, depending on the microfiltration conditions. During frozen storage, cell resistance was also affected by microfiltration conditions, either positively or negatively, compared to centrifugation. The best cryotolerance was obtained for cells microfiltered at a cross-flow velocity of 2 m/s and a transmembrane pressure of 0.15 MPa. This improvement was explained by considering membrane fatty acid composition of Lb. bulgaricus CFL1. This condition increased unsaturated to saturated and cyclic to saturated fatty acid ratios, which enhanced membrane fluidity, thus helping the cells to better resist freezing and frozen storage.     

高密度发酵和真空冷冻干燥工艺对乳酸菌抗冷冻性的影响

经真空冷冻干燥得到的乳酸菌发酵剂存活率和后期的低温贮藏稳定性与诸多因素相关。本文综述了制备乳酸菌发酵剂过程中高密度发酵和真空冷冻干燥工艺的不同对乳酸菌抗冷冻性的影响,其中高密度发酵过程中的培养基组分、培养温度、发酵恒定pH、中和剂的选择、菌体收获时期和发酵结束后处理以及真空冷冻干燥过程中保护剂的添加、预冷冻处理等是影响乳酸菌抗冷冻性的重要因素。通过对这些相关因素的综述分析,为提高乳酸菌发酵剂的冻干存活率和后期的低温贮藏稳定性提供新的思路,且应用抗冷冻性强、活力高的乳酸菌发酵剂对有效提高乳制品的质量和企业的经济效益意义重大。     

Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability.     

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Flow cytometry to assess biochemical pathways in heat-stressed Cronobacter spp. (formerly Enterobacter sakazakii)

Aims: Using a flow cytometry (FC)‐based approach in combination with four selected fluorescent probes, the biochemical pathway activated following the adaptation of Cronobacter spp. to lethal heat stress was investigated. This approach assessed the physiological changes induced in four strains of Cronobacter spp. Methods and Results: Using the commercially available live/dead viability assessment fluorescence probes, live, injured or dead bacterial cells were studied. Cellular respiration and membrane potential were evaluated using the dye‐labelled probe 3,3′‐dihexylocarbocyanine iodide, metabolic activity was evaluated using a fluorescein diacetate (FDA) probe, intracellular pH changes were measured using a carboxy‐fluorescein diacetate succinimidyl ester probe, and reactive oxygen species were measured using a hydroethidine fluorescent probe. Adaptation to lethal heat stress induced physiological changes that potentially improve the survival of Cronobacter spp. Conclusions: These data showed that in situ assessment of physiological behaviour of lethally stressed cells using multiparameter FC is a useful, rapid and sensitive tool to study and assess the viability and physiological state of Cronobacter cells. Significance and Impact of the Study:  This study shows that FC is a valuable tool in the study of physiological aspects of increased survival because of sublethal adaptation to heat.     

Acidification improves cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus CFL1

The effect of acidification of the fermented broth at the end of the culture was examined on the growth and the cryotolerance of Lactobacillus bulgaricus CFL1, as a new means to better preserve lactic acid bacteria. Cryotolerance was investigated by evaluating the loss of specific acidification activity during freezing and frozen storage. An experimental design made it possible to determine optimal acidification conditions that improved cryotolerance, such as pH 5.15 for 30min. These conditions were also conducive to high biomass productivity. By considering the type of acid used, H(2)SO(4) enabled us to obtain cells with better cryotolerance, as compared to HCl. It was also observed that increasing the pH after acidification slightly minimised the acid shock, thus improving cryotolerance. Moreover, it was concluded that this improvement was related to a physiological adaptation of L. bulgaricus CFL1 during the 30-min acidification at pH 5.15.     

Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758

The relationship between lactose starvation and cryotolerance was investigated in Lactobacillus acidophilus RD758. Cryotolerance was measured from the acidification activity of cells recovered after 18-h lactose starvation. It was compared to that of nonstarved cells, both of them in a stationary phase and in the same medium. This measurement allowed quantifying the initial acidification activity before freezing, as well as the loss of acidification activity during freezing and the rate of loss during frozen storage. Even if initial acidification activity was similar for nonstarved and starved bacteria, the latter displayed a significantly better resistance to freezing and frozen storage at -20°C. To investigate the mechanisms that triggered these cryotolerance phenomena, the membrane fatty acid composition was determined by gas chromatography, and the proteome was established by 2-D electrophoresis, for starved and nonstarved cells. The main outcome was that the improved cryotolerance of starved cells was ascribed to two types of physiological responses as a result of starvation. The first one corresponded to an increased synthesis of unsaturated, cyclic, and branched fatty acids, to the detriment of saturated fatty acids, thus corresponding to enhanced membrane fluidity. The second response concerned the upregulation of proteins involved in carbohydrate and energy metabolisms and in pH homeostasis, allowing the cells to be better prepared for counteracting the stress they encountered during subsequent cold stress. These two phenomena led to a cross-protection phenomenon, which allowed better cryotolerance of Lb. acidophilus RD758, following cellular adaptation by starvation.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

Evaluation of lactic acid Streptococci for the preparation of frozen concentrated starter cultures

Eight strains of Streptococcus diacetilactis and Streptococcus lactis were examined for viability, growth rate, lactic acid and diacetyl production in milk and proteolytic activity before and after freezing at --30 degrees C. Concentrations of yeast autolysate, peptone, lactose and citrate as well as the usefulness of milk and whey culture media for active biomass production were investigated. After freezing and storage at --30 degrees C, with the use of non-fat milk as a cryoprotective agent, high survival and endocellular proteolytic activity of the frozen concentrate was achieved. S. diacetilactis sp. and S. lactis 115 were shown to be more biologically active than other strains. Their physiological properties remained unaffected by freezing.     

Cold shock response in Lactococcus lactis ssp. diacetylactis: a comparison of the protection generated by brief pre-treatment at less severe temperatures

Acquired freeze–thaw tolerance was investigated for Lactococcus lactis ssp. diacetylactis. Pre-treatment of microorganisms at less severe temperatures to initiate cold tolerance gave L. lactis ssp. diacetylactis improved cell viability after successive freezings and thawings. The ability of cells to survive freezing–thawing was dependent on factors experienced prior to freezing. Factors affecting lactic acid bacteria survival during freezing–thawing cycles include different diluents, growth phase, and cold temperatures. Viability experiments showed that this strain displaying cold shock cryotolerance had an improved survival capacity in stationary phase. The plasmid contents of lactic acid bacteria isolated from different types, strains DRC-2 and DRC-2C, were examined and compared with the plasmid contents of culture collection strains both before and after cold shock treatment. Using agarose gel electrophoresis, no obvious correlation between the cold shock response and the number of plasmids in the cell could be observed.     

Functional cloning of the dihydropteroate synthase gene of Staphylococcus haemolyticus

Abstract The usefulness of oxonol ( bis -(1,3-dibutylbarbituric acid)trimethine oxonol) as a generally applicable indicator of bacterial viability was investigated using untreated and killed cultures of a variety of bacterial genera. Killing methods involved either heat or bactericidal antibiotics. For all strains tested, the fluorescent dye showed significantly more intense staining of killed than untreated cells. The sensitivity of Aeromonas salmonicida to gentamicin was assessed using oxonol. Although the bacterium was shown to be sensitive to the antibiotic, there was a delay between the time cells lost culturability, as judged by numbers of colony forming units, and that for which a dead cell population could be detected by flow cytometry.     

Effects of protective agents on membrane fluidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

AIMS: To evaluate the effect of protective agents upon survival of Lactobacillus delbrueckii ssp. bulgaricus during freeze-drying and storage, and selective amino acids on cell membrane fluidity. METHODS AND RESULTS: The protective effect of amino-acids and sugars at different concentrations was studied by determining the viability of lyophilized cells after storage under air at 30 degrees C. Survival following freeze-drying was improved by all compounds. During storage, neither proline nor maltose had protective effects on lyophilized Lact. delbrueckii ssp. bulgaricus. Glutamate 5% and aspartate 5% showed similar protection capability during freeze-drying (94-95%) and after storage (92-99%). Fluorescence probes (DPH and TMA-DPH) were used to study the effect of both amino acids on membrane fluidity. Polarization decreased with increasing concentrations of glutamate or aspartate. Lowest values were observed with TMA-DPH. CONCLUSIONS: Glutamate 5% and aspartate 5% allowed maintaining high viability rates during freeze-drying and storage of Lact. delbrueckii ssp. bulgaricus because of an increase of the membrane fluidity by inserting in the interfacial region of bacterial plasma membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show the first evidence of the mechanisms underlying glutamate and aspartate as lyoprotectors.     

Multiparametric flow cytometry and cell sorting for the assessment of viable,injured, and dead bifidobacterium cells during bile salt stress

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.     

Long-Term Storage of Bacteriophages of Lactic Streptococci

Four phage strains representing phages of Streptococcus lactis, S. cremoris, and S. diacetilactis were selected for the observation of the effect of cold storage on their viability. Phages were stored at 4 C and at -18 C, or were frozen at approximately -70 C and stored at -18 C. They were found to display a high degree of stability with these storage methods. The same phage strains showed good stability to storage at room temperature for 3 weeks after thawing and also to alternate freezing and thawing eight times. Three series consisting of from 23 to 31 lactic streptococcal phage preparations were observed over periods extending up to 6 years, and with only a few exceptions were found to store satisfactorily at -18 C after quick freezing. Although the same phage preparations stored at 4 C were generally somewhat less stable, many were stable when stored by both methods.     

Subcellular membrane fluidity of Lactobacillus delbrueckii subsp. bulgaricus under cold and osmotic stress

Cryopreservation of lactic acid bacteria may lead to undesirable cell death and functionality losses. The membrane is the first target for cell injury and plays a key role in bacterial cryotolerance. This work aimed at investigating at a subcellular resolution the membrane fluidity of two populations of Lactobacillus delbrueckii subsp. bulgaricus when subjected to cold and osmotic stresses associated to freezing. Cells were cultivated at 42 °C in mild whey medium, and they were exposed to sucrose solutions of different osmolarities (300 and 1800 mOsm L−1) after harvest. Synchrotron fluorescence microscopy was used to measure membrane fluidity of cells labeled with the cytoplasmic membrane probe 1-[4 (trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Images were acquired at 25 and 0 °C, and more than a thousand cells were individually analyzed. Results revealed that a bacterial population characterized by high membrane fluidity and a homogeneous distribution of fluidity values appeared to be positively related to freeze-thaw resistance. Furthermore, rigid domains with different anisotropy values were observed and the occurrence of these domains was more important in the freeze-sensitive bacterial population. The freeze-sensitive cells exhibited a broadening of existing highly rigid lipid domains with osmotic stress. The enlargement of domains might be ascribed to the interaction of sucrose with membrane phospholipids, leading to membrane disorganization and cell degradation.     

Survival and Activity of Frozen Starter Cultures for Cheese Manufacture

A study has been conducted on the effect of freezing and storage in liquid nitrogen on 13 strains of lactic streptococci. Cultures were frozen in droplet form and collected in mesh bags. After rapid thawing, the activity of the frozen cultures was compared with a culture of the same organism of the age usually used in cheese-making. The activities of the test and control cultures were traced simultaneously by continuous recording of the pH changes in inoculated milks. Viable counts were performed before and after freezing in liquid nitrogen and after storage in liquid nitrogen. There was no decrease in viable count or loss in activity of the cultures due to freezing and storage. Frozen cultures of some strains showed a shorter lag period after inoculation of milk than control cultures. Frozen concentrated cheese-starter cultures behaved normally in the manufacture of Cheddar cheese.     

Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC₄(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.     

Identification of a Cold Shock Gene in Lactic Acid Bacteria and the Effect of Cold Shock on Cryotolerance

When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested. Received: 18 October 1996 / Accepted: 28 January 1997