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.     

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.     

Dynamic analysis of <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis> CFL1 physiological characteristics during fermentation

This study aimed at examining and comparing the relevance of various methods in order to discriminate different cellular states of Lactobacillus bulgaricus CFL1 and to improve knowledge on the dynamics of the cellular physiological state during growth and acidification. By using four fluorescent probes combined with multiparametric flow cytometry, membrane integrity, intracellular esterase activity, cellular vitality, membrane depolarization, and intracellular pH were quantified throughout fermentations. Results were compared and correlated with measurements of cultivability, acidification activity (Cinac system), and cellular ability to recover growth in fresh medium (Bioscreen system). The Cinac system and flow cytometry were relevant to distinguish different physiological states throughout growth. Lb. bulgaricus cells maintained their high viability, energetic state, membrane potential, and pH gradient in the late stationary phase, despite the gradual decrease of both cultivability and acidification activity. Viability and membrane integrity were maintained during acidification, at the expense of their cultivability and acidification activity. Finally, this study demonstrated that the physiological state during fermentation was strongly affected by intracellular pH and the pH gradient. The critical pHi of Lb. bulgaricus CFL1 was found to be equal to pH 5.8. Through linear relationships between dpH and cultivability and pHi and acidification activity, pHi and dpH well described the time course of metabolic activity, cultivability, and viability in a single analysis.     

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.     

交互保护对干酪乳杆菌ATCC 393~(TM)存活的影响

【目的】以干酪乳杆菌典型株ATCC 393TM(Lactobacillus casei ATCC 393TM)为实验菌株,研究其在多重胁迫环境下的交互保护应答机制。【方法】比较不同亚适应条件(热、H2O2、酸、胆盐)处理后菌体细胞在热致死条件(60℃)及氧致死条件H2O2(5mmol/L)下的存活率变化,并集中考察了最佳亚适应条件-酸适应的不同处理方式对细胞交互保护存活率、胞内pH以及脂肪酸含量的影响。【结果】交互保护对干酪乳杆菌ATCC393生理活性的影响因亚适应及致死条件而异:酸胁迫预适应能够显著提高细胞的交互胁迫抗性,其中,盐酸预适应的交互保护效果优于乳酸,其预适应引发的生理应答效应使细胞在应对热致死和氧致死胁迫时存活率分别提高了305倍和173倍;进一步的研究表明,酸预适应提高细胞存活率的作用机制可能与其能够显著改善胁迫环境下的胞内pH和细胞膜脂肪酸不饱和度相关。【结论】盐酸预适应对干酪乳杆菌典型株ATCC393的交互保护作用最为显著,并能够维持胁迫条件下细胞生理状态的相对稳定,本研究将有助于进一步解析干酪乳杆菌在对抗不同胁迫环境的过程中生理应答机制间的相互作用关系。     

Degradation of milk‐based bioactive peptides by yogurt fermentation bacteria*

Aims: To analyse the effect of cell‐associated peptidases in yogurt starter culture strains Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST) on milk‐protein‐based antimicrobial and hypotensive peptides in order to determine their survival in yogurt‐type dairy foods. Methods and Results: The 11mer antimicrobial and 12mer hypotensive milk‐protein‐derived peptides were incubated with mid‐log cells of LB and ST, which are required for yogurt production. Incubations were performed at pH 4·5 and 7·0, and samples removed at various time points were analysed by reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The peptides remained mostly intact at pH 4·5 in the presence of ST strains and moderately digested by exposure to LB cells. Peptide loss occurred more rapidly and was more extensive after incubation at pH 7·0. Conclusions: The 11mer and 12mer bioactive peptides may be added at the end of the yogurt‐making process when the pH level has dropped to 4·5, limiting the overall extent of proteolysis. Significance and Impact of the Study: The results show the feasibility of using milk‐protein‐based antimicrobial and hypotensive peptides as food supplements to improve the health‐promoting qualities of liquid and semi‐solid dairy foods prepared by the yogurt fermentation process.     

Development of a structured model for batch cultures of lactic acid bacteria

A combined stochastic-deterministic model able to predict the growth curve of microorganisms, from inoculation to death, is presented. The proposed model is based on the assumption that microorganisms can experience two different physiological states: non-proliferating and proliferating. The former being the physiological state of the cells right after their inoculation into the new extracellular environment; the latter the state of microorganisms after adaptation to the new medium. To validate the model, a Lactobacillus bulgaricus strain was tested in a medium at pH 4.6 at two different temperatures (42°C and 35°C). Curves representing the bacterial growth cycle were satisfactorily fitted by means of the proposed model. Moreover, due to the mechanistic structure of the proposed model, valuable quantitative information on the following was obtained: rate of conversion of non-proliferating cells into proliferating cells, growth and death rate of proliferating cells, and rate of nutrient consumption.     

Bioconversion of isoflavone glycosides to aglycones,mineral bioavailability and vitamin B complex in fermented soymilk by probiotic bacteria and yeast

Aim: To study the role of β‐glucosidase producing probiotic bacteria and yeast in the biotransformation of isoflavone glycosides to aglycones, mineral bioavailability and vitamin B complex in fermented soymilk. Methods and Results: Five isolates of probiotic lactic acid bacteria (LAB), Lactobacillus acidophilus B4496, Lactobacillus bulgaricus CFR2028, Lactobacillus casei B1922, Lactobacillus plantarum B4495 and Lactobacillus fermentum B4655 with yeast Saccharomyces boulardii were used to ferment soymilk to obtain the bioactive isoflavones, genistein and daidzein. High‐performance liquid chromatography was used to analyse the concentration of isoflavones. Bioactive aglycones genistein and daidzein after 24 and 48 h of fermentation ranged from 97·49 to 98·49% and 62·71 to 92·31% respectively with different combinations of LAB with yeast. Increase in bioavailability of minerals and vitamin B complex were also observed in fermented soymilk. Conclusions: LAB in combination with yeast S. boulardii has great potential for the enrichment of bioactive isoflavones, enhancing the viability of LAB strains, decreasing the antinutrient phytic acid and increasing the mineral bioavailability in soymilk fermentation. Significance and Impact of the Study: Fermentation of soymilk with probiotic organisms improves the bioavailability of isoflavones, assists in digestion of protein, provides more soluble calcium, enhances intestinal health and supports immune system. Increased isoflavone aglycone content in fermented soymilk improves the biological functionality of soymilk.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.     

Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres

The viability of lyophilized cultures of Lactobacillus bulgaricus in skim milk, during storage at different temperatures, relative humidities, and atmospheres was investigated. Survival was greatest at 11% relative humidity and at 5°C. Indirect and direct evidence is presented supporting the hypothesis that membrane damage occurs during storage. Experiments on the lipid composition of the cell membrane demonstrate that changes occur with time that are probably the result of oxidation. A study on the lipid composition of the cell membrane by gas chromatography showed that the unsaturated/saturated fatty acid index changes with time during storage.     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

Changes in the cell membrane of Lactobacillus bulgaricus during storage following freeze-drying

Summary The mechanism of inactivation of freeze-dried Lactobacillus bulgaricus during storage in maltodextrin under controlled humidity was investigated. Evidence is presented supporting the hypothesis that membrane damage occurs during storage. A study on the lipid composition of the cells by gas chromatography showed a decrease in the unsaturated and saturated fatty acid content of the cell. Further evidence indicating membrane damage includes a decrease in membrane bound proton-translocating ATPase activity.     

Modelling the acidifying activity profile of Lactobacillus bulgaricus cultures

A model that fully describes the typical pH(t) profile representing the lactic acid production kinetics of Lactobacillus bulgaricus cultures is reported. The model, a four-parameter function [pH = (A–D)/(1 + (t/C)^B) + D], is able to fit any change on the experimental pH-time curves, due to variations on the inoculum cell concentration of the culture. The four fitting parameters(A, B, C and D) of this model are closely related to the lactic acid fermentation and they have a physical meaning. Parameters A and D represent the initial and final pH of the culture, respectively. Parameter B is related to the slope of the linear decreasing region from the pH-time curve and C represents the time at which half of the total decrement of pH is achieved. The proposed model can be used not only for evaluating and comparing the acidifying capacity of homolactic cultures but also for predictions of final fermentation times.     

Multistrain probiotic production by co‐culture fermentation in a lab‐scale bioreactor

Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.     

Fermentation pH Influences the Physiological-State Dynamics of Lactobacillus bulgaricus CFL1 during pH-Controlled Culture

This study aims at better understanding the effects of fermentation pH and harvesting time on Lactobacillus bulgaricus CFL1 cellular state in order to improve knowledge of the dynamics of the physiological state and to better manage starter production. The Cinac system and multiparametric flow cytometry were used to characterize and compare the progress of the physiological events that occurred during pH 6 and pH 5 controlled cultures. Acidification activity, membrane damage, enzymatic activity, cellular depolarization, intracellular pH, and pH gradient were determined and compared during growing conditions. Strong differences in the time course of viability, membrane integrity, and acidification activity were displayed between pH 6 and pH 5 cultures. As a main result, the pH 5 control during fermentation allowed the cells to maintain a more robust physiological state, with high viability and stable acidification activity throughout growth, in opposition to a viability decrease and fluctuation of activity at pH 6. This result was mainly explained by differences in lactate concentration in the culture medium and in pH gradient value. The elevated content of the ionic lactate form at high pH values damaged membrane integrity that led to a viability decrease. In contrast, the high pH gradient observed throughout pH 5 cultures was associated with an increased energetic level that helped the cells maintain their physiological state. Such results may benefit industrial starter producers and fermented-product manufacturers by allowing them to better control the quality of their starters, before freezing or before using them for food fermentation.Lactic acid bacteria are traditionally used to produce or to preserve various food products such as fermented milks, meats, and vegetables. Their ability to initiate rapid acidification of the raw material is essential to improve the flavor, texture, and safety of these products (11, 14). In order to prevent poor fermentation yields and to improve the quality and reliability of the products, it is important to maintain proper control starter production. This control may be achieved by studying the effects of process parameters on the growth kinetics of the bacteria and on their acidification activity and physiological state in growing conditions. Among all process parameters, pH and harvesting time are key factors that strongly influence the physiological state of lactic acid bacteria after fermentation and stabilization.Lactic acid starters are currently produced using pH-controlled pure cultures (6), during which pH is generally regulated at an optimal value by continuously adding sodium hydroxide or ammonia in the bioreactor (23). Various growth characteristics such as maximal biomass concentration, specific growth rate, fermentation time, sugar consumption or growth, and product yields are significantly influenced by the pH control value (1, 4). Optimal pH ranges were therefore determined for several lactic acid bacteria, such as Streptococcus thermophilus (pH 6.5), Lactobacillus bulgaricus (pH 5.8 to 6) (5, 22), or Lactococcus lactis subsp. cremoris (pH 6.3 to 6.9) (8).Compared to acidic fermentations, pH-controlled cultures led to higher growth yields and productivity (9, 23) as a result of the lower level of nondissociated lactic acid in the culture medium (2, 12, 15). The acidification of the cytoplasm induced by the nondissociated form of the weak organic acid leads to the collapse of the proton motive force (13). This phenomenon inhibits nutrient transport and enzymatic reactions and leads to DNA alteration and biomass inactivation (12). Maintaining the extracellular pH (pHext) at a high value helps the cells stabilize their intracellular pH at a sufficiently high value (9), thus decreasing the inhibiting effect of lactic acid.Fermentation pH also acts on energetic parameters, such as internal pH (pHi), pH gradient (dpH), proton motive force, membrane potential, NADH/NAD ratio, ATP level and rate of ATP formation, and lactate dehydrogenase and ATPase activity (1, 9, 17). During batch cultures of L. lactis performed with or without pH control, Cachon et al. (9) showed that pH control has a significant influence on the variations of pHi, dpH, and NADH/NAD ratio, thus acting on growth parameters. Moreover, in batch cultures, pHi is dependent upon both the external pH and the age of culture. Mercade et al. (17) showed that cultures of L. bulgaricus at controlled pH 6.4 are inhibited at the level of anabolism but were not energy limited. They are characterized by a high maintenance coefficient in contrast to cultures without pH control which consume intracellular energy for pHi regulation.The effect of pH on cellular physiology is confirmed by other studies which show that it influences acidification activity of lactic acid bacteria (23-25). Whereas Wang et al. (25) indicated that Lactobacillus acidophilus cells grown at optimal pH display a higher residual acidification activity than cells grown at lower pH control values, Schepers et al. (24) and Savoie et al. (23) demonstrated that this activity is higher when starters are produced without pH control or at low pH control values. These authors explained that conditions generating high biomass concentrations do not systematically lead to cells with an efficient acidification activity.From this information, the effect of pH control was elucidated on growth and energetic parameters, whereas its effect on the dynamic of cellular physiology, viability, and acidification activity during growth is still not determined.A few authors demonstrated that the harvesting time has a strong impact on cellular parameters such as viability and acidification activity (3, 20, 24). Béal et al. (6) specified that there is an optimal range of time during which to harvest cells in a good physiological state, i.e., at a high cellular concentration and a high acidification activity. However, since this optimal range is strongly strain and condition dependent, more information is needed about the influence of harvesting time on physiological parameters.In order to improve knowledge about the effects of fermentation pH and harvesting time on starter''s quality, we sought here to apply some rapid and relevant methods to characterize the dynamic of L. delbrueckii subsp. bulgaricus CFL1 physiological state throughout pH 6 and pH 5 fermentations. This might allow industrial starter producers to better control their fermentations and to achieve high-quality starters. Among the available methods, the Cinac system and multiparametric flow cytometry, associated with plate counts, made it possible to determine and compare different physiological parameters such as cultivability, acidification activity (Cinac system), membrane damage, enzymatic activity, cell depolarization, intracellular pH, and pH gradient (flow cytometry) (20). Two dynamic schemes of the time course of the physiological state during pH 6 or pH 5 cultures are proposed and discussed.     

Ethanol addition enhances acid treatment to eliminate Lactobacillus fermentum from the fermentation process for fuel ethanol production

Fermentation is one of the most critical steps of the fuel ethanol production and it is directly influenced by the fermentation system, selected yeast, and bacterial contamination, especially from the genus Lactobacillus. To control the contamination, the industry applies antibiotics and biocides; however, these substances can result in an increased cost and environmental problems. The use of the acid treatment of cells (water‐diluted sulphuric acid, adjusted to pH 2·0–2·5) between the fermentation cycles is not always effective to combat the bacterial contamination. In this context, this study aimed to evaluate the effect of ethanol addition to the acid treatment to control the bacterial growth in a fed‐batch system with cell recycling, using the industrial yeast strain Saccharomyces cerevisiae PE–2. When only the acid treatment was used, the population of Lactobacillus fermentum had a 3‐log reduction at the end of the sixth fermentation cycle; however, when 5% of ethanol was added to the acid solution, the viability of the bacterium was completely lost even after the first round of cell treatment. The acid treatment +5% ethanol was able to kill L. fermentum cells without affecting the ethanol yield and with a low residual sugar concentration in the fermented must.

Significance and Impact of the Study

In Brazilian ethanol‐producing industry, water‐diluted sulphuric acid is used to treat the cell mass at low pH (2·0) between the fermentative cycles. This procedure reduces the number of Lactobacillus fermentum from 10⁷ to 10⁴ CFU per ml. However, the addition of 5% ethanol to the acid treatment causes the complete loss of bacterial cell viability in fed‐batch fermentation with six cell recycles. The ethanol yield and yeast cell viability are not affected. These data indicate the feasibility of adding ethanol to the acid solution replacing the antibiotic use, offering a low cost and a low amount of residue in the biomass.     

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     

Optimization of Exopolysaccharide Production by Lactobacillus delbrueckii subsp. bulgaricus RR Grown in a Semidefined Medium

The optimal fermentation temperature, pH, and Bacto-casitone (Difco Laboratories, Detroit, Mich.) concentration for production of exopolysaccharide by Lactobacillus delbrueckii subsp. bulgaricus RR in a semidefined medium were determined by using response surface methods. The design consisted of 20 experiments, 15 unique combinations, and five replications. All fermentations were conducted in a fermentor with a 2.5-liter working volume and were terminated when 90% of the glucose in the medium had been consumed. The population of L. delbrueckii subsp. bulgaricus RR and exopolysaccharide content were measured at the end of each fermentation. The optimum temperature, pH, and Bacto-casitone concentration for exopolysaccharide production were 38°C, 5, and 30 g/liter, respectively, with a predicted yield of 295 mg of exopolysaccharide/liter. The actual yield under these conditions was 354 mg of exopolysaccharide/liter, which was within the 95% confidence interval (217 to 374 mg of exopolysaccharide/liter). An additional experiment conducted under optimum conditions showed that exopolysaccharide production was growth associated, with a specific production at the endpoint of 101.4 mg/g of dry cells. Finally, to obtain material for further characterization, a 100-liter fermentation was conducted under optimum conditions. Twenty-nine grams of exopolysaccharide was isolated from centrifuged, ultrafiltered fermentation broth by ethanol precipitation.