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.     

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.     

Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.     

Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758

The effect of different cooling temperatures and durations on resistance to freezing and to frozen storage at -20 degrees C in Lactobacillus acidophilus RD758 was studied, by using a central composite rotatable design. A cold adaptation was observed when the cells were maintained at moderate temperature (26 degrees C) for a long time (8h) before being cooled to the final temperature of 15 degrees C. These conditions led to a low rate of loss in acidification activity during frozen storage (0.64 minday(-1)) and a high residual acidification activity after 180 days of frozen storage (1011 min). The experimental design allowed us to determine optimal cooling conditions, which were established at 28 degrees C during 8h. Adaptation to cold temperatures was related to an increase in the unsaturated to saturated fatty acid ratio and in the relative cycC19:0 fatty acid concentration. Moreover, an increased synthesis of four specific proteins was observed as an adaptive response to the optimal cooling conditions. They included the stress protein ATP-dependent ClpP and two cold induced proteins: pyruvate kinase and a putative glycoprotein endopeptidase.     

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.     

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.     

Studies on the effects of low temperatures on lactic acid bacteria

Studies were conducted on different strains of L. bulgaricus, L. casei, S. thermophilus, S. lactis, and S. cremoris isolated in Bulgaria and applied as pure cultures and in combinations as starters. All the strains under investigation were found to preserve, on “freezing-thawing” their characteristic morphological and biochemical properties, regardless of the temperature and rate of cooling, but the optimum freezing temperature of the strains studied is ?196 °C (in liquid nitrogen). High cooling rates provide higher viability and activity of lactic acid bacterial cells. Lactic acid streptococci, S. lactis and S. thermophilus, are considerably more resistant than lactic acid rods, L. casei and L. bulgaricus, at all the freezing regimens tested.     

乳酸菌的抗冷冻性及耐受机理

乳酸菌发酵剂在工业生产过程中,会受到冷冻的刺激,如真空冷冻干燥及后期的低温保藏,此外,发酵乳制品的保藏和干酪的成熟过程也都在低温中进行。这些均会对乳酸菌发酵剂及发酵乳制品质量产生一定的影响。因此,掌握乳酸菌在冷冻条件下的反应机理有助于优化发酵剂和发酵乳制品在工业生产中的冷冻、发酵和贮藏条件,从而提高产品质量和生产效益。本文对乳酸菌的抗冷冻性及机理进行了分析,并对发酵剂的保护提出具体措施。     

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°C and −20°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°C and −80°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°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.     

Production of D-tagatose, a low caloric sweetener during milk fermentation using L-arabinose isomerase

Lactobacillusdelbrueckii subsp. bulgaricus and Streptococcus thermophilus are used for the biotransformation of milk in yoghurt. During milk fermentation, these lactic acid bacteria (LAB) hydrolyze lactose producing a glucose moiety that is further metabolized and a galactose moiety that they are enable to metabolize. We investigated the ability of L. bulgaricus and S. thermophilus strains expressing a heterologous L-arabinose isomerase to convert residual D-galactose to D-tagatose. The Bacillus stearothermophilus US100l-arabinose isomerase (US100l-AI) was expressed in both LAB, using a new shuttle vector where the araA US100 gene is under the control of the strong and constitutive promoter of the L. bulgaricus ATCC 11842 hlbA gene. The production of L-AI by these LAB allowed the bioconversion of D-galactose to D-tagatose during fermentation in laboratory media and milk. We also established that the addition of L-AI to milk also allowed the conversion of D-galactose into D-tagatose during the fermentation process.     

Induction of 2'-5' A synthetase activity and interferon in humans by bacteria used in dairy products.

The production of interferon by fasted human subjects in response to lactic bacteria Lactobacillus bulgaricus and Streptococcus thermophilus was evaluated in vivo and in vitro. The 2'-5' A synthetase activity of blood mononuclear cells was used to estimate interferon production following a single ingestion of 10(11) bacteria in yoghurt or sterile milk (controls). The level of the 2'-5' A synthetase of the yoghurt fed subjects was 83% (p = 0.002) higher than that of the milk fed controls 24 hours after ingestion. The baseline value remained unchanged in the control group. Blood mononuclear cells from a second group of subjects, were cultured with lactic bacteria for 48 hours, their cell-free supernatants contained gamma interferon. These results suggest that a transient production of interferon can be induced in healthy subjects by the lactic bacteria used in food processing.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

Survival kinetics of lactic acid starter cultures during and after freeze drying

Survival kinetics of lactic acid starter cultures were modeled considering the microorganism and external medium interfacial area as the critical factors determining the resistance of the microorganisms to freeze-drying. Surviving fraction of the microorganisms increased with the increasing biomass concentration during freeze-drying, and this is attributed to the mutual shielding effect of the microorganisms against the severe conditions of the external medium. Survival of the microorganisms over the storage period after freeze drying was enhanced by the presence of dead microorganisms which reduce the interfacial area between the live cells and the external medium. Streptococcus thermophilus was found to be more resistant to freeze-drying conditions than Lactobacillus bulgaricus. Storage under vacuum or nitrogen was superior to storage under air. Poor survival rates under air was attributed to oxygen diffusion into the dry cells through the interfacial area.     

Classification of lactic acid bacteria with UV-resonance Raman spectroscopy

UV-resonance Raman spectroscopy is applied as a method for the identification of lactic acid bacteria from yogurt. Eight different strains of bacteria from Lactobacillus acidophilus, L. delbrueckii ssp. bulgaricus, and Streptococcus thermophilus were investigated. At an excitation wavelength of 244 nm signals from nucleic acids and proteins are selectively enhanced. Classification was accomplished using different chemometric methods. In a first attempt, the unsupervised methods hierarchical cluster analysis and principal component analysis were applied to investigate natural grouping in the data. In a second step the spectra were analyzed using several supervised methods: K-nearest neighbor classifier, nearest mean classifier, linear discriminant analysis, and support vector machines.     

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.