Influence of temperature and membrane lipid composition on the osmotic water permeability of teleost gills.

Osmotic water uptake was measured gravimetrically in isolated, ligated gill arches from trout (acclimated to and incubated at 5 degrees and 20 degrees C) and tilapia (21.5 degrees and 33 degrees C). For both species, incubation of arches at the higher temperature led to 1.5- to 3-fold greater measures of water weight gain. However, gills from warmer-acclimated trout and tilapia had 1- to >3-fold lower the initial rate and 1.5- to >2.5-fold lower the extent of water uptake seen in colder-acclimated conspecifics. Both the incubation temperature sensitivity and the acclimation effects are consistent with transmembrane water permeation. Calcium-free incubations (permitting paracellular water movement) also indicated that interfacial cell membranes contribute to gill permeability characteristics; without calcium, trout gill osmotic water uptake values increased 1.5- to 2-fold, and the temperature dependence of water uptake decreased (initial rate) or was eliminated (extent). The specific contribution of cholesterol to restricting barrier membrane water permeability was indicated by concentration-dependent increases in water uptake in the presence of either nystatin (a cholesterol-complexing, pore-forming agent) or methyl-beta-cyclodextrin (which selectively depletes membrane cholesterol). In addition, a cholesterol-specific cytochemical probe (filipin) intensely labeled the apical surface membranes of trout and tilapia gill epithelium. In summary, these studies implicate membrane cholesterol in determining water permeability in fish gills.     

Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus

In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared. Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids. This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio. In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined. From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified. The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization. The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes.     

Influence of pH, temperature, and inoculum composition on mixed cultures of Streptococcus thermophilus 404 and Lactobacillus bulgaricus 398

The effects of pH, temperature, and inoculum composition on Streptococcus thermophilus 404 and Lactobacillus bulgaricus 398 mixed cultures were studied. Linear quadratic models according to these three variables were proposed to express the growth and acidification characteristics, as well as the final proportion between the two strains. Initial and operating conditions, allowing maximization of these characteristics were determined. Finally, a graphic method to predict both final total concentration and final proportion of the two bacteria as a function of pH, temperature and inoculum composition is presented.     

Influence of Calcium and Manganese on Dechaining of Lactobacillus bulgaricus

The events responsible for the transition of Lactobacillus bulgaricus 1243-F from long filamentous chains to short bacilloid rods were examined in a cation-depleted liquid medium. In the presence of magnesium only, cells grew as long chains of unseparated cells. The addition of 100 μM to 1 mM calcium or manganese to this medium resulted in the dechaining of these cells to short bacilloid rods. Fe²⁺, Zn²⁺, Co²⁺, and Cu²⁺ failed to induce dechaining. Induction of calcium and manganese dechaining functioned under controlled pH maintained at 5.0 and 6.0 but not at pH 7.0. This was consistent with a previous report showing failure in synthesis of dechaining enzymes by L. bulgaricus under pH conditions approaching alkalinity (S. K. Rhee and M. Y. Pack, J. Bacteriol. 144:865-868, 1980). We conclude that under pH conditions which permit synthesis of dechaining enzymes, calcium and manganese are necessary for dechaining activity.     

Membrane lipid composition, fluidity, and surface charge changes in response to growth of the fresh water cyanobacterium Synechococcus 6311 under high salinity

The effect of adaptation to saline growth of a fresh water cyanobacterium Synechococcus 6311 on components of the cytoplasmic membranes and thylakoids was investigated. Significant changes in membrane surface charge, lipid, fatty acid, and carotenoid composition were observed upon transfer of the cells from a low salt (0.015 M NaCl) to a high salt (0.50 M NaCl) growth medium. Very similar changes in the polar lipid classes and fatty acid composition were observed in both membranes, but changes in fluidity and surface charge and a significant shift in the protein to lipid ratio were only apparent in the cytoplasmic membranes. The fluidity and surface charge data correlate well with functional studies and we can attribute the cytoplasmic membrane as the major site of interaction and adaptation to the saline environment.     

Determination of cells' water membrane permeability: unexpected high osmotic permeability of Saccharomyces cerevisiae

Water permeability (Lp), calculated from the volume variations of cells subjected to an osmotic shock, is classically used to characterize cell membrane properties. In this work, we have shown the importance of the kind of mixing reactor used to measure the Lp parameter. A mathematical model including the mixing time constant has been proposed allowing an accurate Lp estimation even though the mixing time constant is higher than the cell time constant obtained in response to a perfect shock. The estimated Lp values of human leukemia K562 cells were found to be the same whatever the mixing time constant. The Lp value of Saccharomyces cerevisiae could not be exactly estimated. However, S. cerevisiae has unexpectedly high water permeability, implying that this yeast may contain water channels in the membrane. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 62-70, 1997.     

Influence of medium composition, pH and temperature on the growth and viability of Lactobacillus acidophilus

The Lactobacillus acidophilus growth was investigated to find the optimal concentrations of 2 nitrogen sources (thought to be in excess) and 2 organic acids. A Plackctt and Burman experimental design was used allowing identification of the more important factors with very few experiments. In the studied range, all the factors had a linear effect and a great influence on the final viability. 20 g/L of each nitrogen sources, 3 g sodium-citrate/L and 5 g sodium acetate/L are necessary. pH, temperature and glucose had poor influence.     

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.     

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.     

Effect of sugars and growth media on the dehydration of Lactobacillus delbrueckii ssp. bulgaricus

AIMS: The efficiency of trehalose, sucrose and maltose to protect Lactobacillus bulgaricus during drying has been evaluated in bacteria grown at low water activity. METHODS AND RESULTS: Bacteria were grown in MRS (control), and in MRS supplemented with sucrose (MRS-sucrose) or with polyethyleneglycol (PEG) (MRS-PEG) as low water activity media. The growth in low water activity media (MRS-sucrose and MRS-PEG) prior to drying enhanced the effectiveness of trehalose as thermoprotectant during drying. The efficiency of sucrose was improved when bacteria were grown in MRS-sucrose. On the other hand, the growth in both low water activity media did not affect the efficiency of maltose. The damage produced during dehydration has been evaluated by means of growth kinetics in milk. The preservation of bacteria dehydrated with sucrose, after growing them in MRS-sucrose, appears to be as efficient as the dehydration with trehalose. CONCLUSIONS: The growth of L. bulgaricus in low water activity media enhances the protective action of trehalose and sucrose. SIGNIFICANCE AND IMPACT OF THE THE STUDY: These results may aid the dairy industry to improve the recovery of the starters at low costs after preservation processes.     

Influence of controlled pH and temperature on the growth and acidification of pure cultures of Streptococcus thermophilus 404 and Lactobacillus bulgaricus 398

Summary The effect of pH and temperature on the growth and acidification characteristics of Streptococcus thermophilus 404 and Lactobacillus bulgaricus 398 were studied in order to optimize starter production. A quadratic two-variable model for each of these parameters is proposed. Optimal growth conditions were found at pH 6.5 and 40° C for S. thermophilus and pH 5.8 and 44° C for L. bulgaricus. Maximum acidification was obtained at pH values and temperatures higher than the optimal growth conditions. In addition, the two strains were generally more sensitive to pH effect.Offprint requests to: C. Beal     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

发酵中含氮类原料对保加利亚乳杆菌活菌数的影响

目的研究生产发酵过程中培养基含氮原料与发酵的相关性。方法实验通过对不同生产厂家三种原料进行理化指标检测,并通过正交试验,比较不同原料发酵后保加利亚乳杆菌发酵液活菌数。结果最佳条件为酪蛋白胨氨基氮含量高,酵母浸粉的炽灼残渣低,牛肉粉总氮含量高。结论经过相同原料不同生产厂家的配方发酵后的发酵液活菌数存在差异,其理化指标对发酵有一定影响。     

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

     

The influence of lipid composition on glycophorin-induced bilayer permeability

Glycophorin was incorporated into large unilamellar vesicles and the bilayer permeability was measured as a function of the lipid composition. In agreement with previous data (Van der Steen, A.T.M., De Kruijff, B. and De Gier, J. (1982) Biochim. Biophys. Acta 691, 13-23) it was found that glycophorin greatly increased the bilayer permeability of DOPC vesicles. This effect was observed for a large variety of phosphatidylcholines, differing in their fatty acid composition and homogeneity. In sharp contrast, it was observed that variations in the polar headgroups by incorporation of DOPE, DOPS and, to a lesser extent, cholesterol, into the DOPC/glycophorin vesicles restored the barrier function. These results are compared to the size of the particles, revealed by freeze-fracture electron microscopy on the glycophorin-containing bilayer and are discussed in the light of various types of lipid-protein interactions and protein aggregation state.