Bifidobacteria Strain Behavior Toward Cholesterol: Coprecipitation with Bile Salts and Assimilation

Resting cells and growing cells of bifidobacteria strains exhibited an ability to remove cholesterol in the presence of bile salts. In resting cell assays, the removed cholesterol was precipitated in the presence of cholic acid at pH values lower than 5.4. However, this precipitated cholesterol was redissolved when the pellets were washed with phosphate buffer, pH 7, and no cholesterol was found in the cells. It appears that this precipitation is a transient phenomenon. In the case of growing cells, the removed cholesterol was partially recovered when cells were washed with phosphate buffer, pH 7, while the remaining cholesterol was extracted from the cells. Cultured in the presence of radiolabeled free or esterified cholesterol, bifidobacteria strains were able to assimilate esterified cholesterol. It is concluded that the removal of cholesterol from the growth medium by bifidobacteria strains is due to both bacterial assimilation and precipitation of cholesterol. Received: 8 February 1996/Accepted: 11 March 1996     

Bile salt hydrolase and cholesterol removal effect by Bifidobacterium bifidum NRRL 1976

Summary Growing cells of Bifidobacterum bifidum NRRL 1976 exhibited an ability to remove cholesterol in the presence of bile salts. The cholesterol removal by Bifidobacterium bifidum was due to a co-precipitation together with unconjugated bile acids, which was linked to the bile salt hydrolase (BSH) activity of the cells at pH values lower than 5.0 and the cholesterol removed was partially recovered when the cells were washed with phosphate buffer at pH 7, while the remaining cholesterol was extracted from the cells. It is concluded that the removal of cholesterol from the growth medium by Bifidobacterium bifidum strain is due to both bacterial assimilation and precipitation of cholesterol.     

Endospore formation by Streptomyces avermitilis in submerged culture

The capability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycles on synthetic medium CP1 with glucose under phosphate limitation, and (2) in autolysing cell suspensions of high density obtained by tenfold concentration of a stationary-phase culture grown in a synthetic medium resuspended in phosphate buffer (pH 7.2) with 0.2% CaCl2. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to pertain to the main species-defining features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in a surface culture.     

The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity.

To study the mechanism of the propsed assimilation of cholesterol, we cultured various strains of Lactobacillus acidophilus and a Bifidobacterium sp. in the presence of cholesterol and oxgall. During culturing, both cholesterol and bile salts were precipitated. Because of bacterial bile salt deconjugation, no conjugated bile salts were observed in either the culture fluids or the pellets. During incubation, the cell count and optical density decreased. The degree of precipitation of bile salts and of cholesterol was dependent on the culture conditions. If L. acidophilus RP32 was cultured under acidifying conditions, the degree of precipitation of deconjugated bile salts was higher than if the pH was maintained at 6.0. Under acidifying conditions, cholesterol was coprecipitated with the bile salts, whereas in pH-controlled cultures, no coprecipitation of cholesterol was observed. From control experiments with different mixtures of bile salts, it appeared that coprecipitation of cholesterol during culturing was a result of formation of deconjugated bile salts, which have a decreased solubility at pH values lower than 6.0. It is concluded that the removal of cholesterol from the culture medium by L. acidophilus RP32 and other species is not due to bacterial uptake of cholesterol, but results from bacterial bile salt-deconjugating activity.     

Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

Effect of pH alteration on growth and glucose oxidation in myoblast cultures

The growth of chick heart cells in culture declines when the cells reach confluency. The decline in growth rate is associated with both a decrease in the pH of the bicarbonate-CO₂ buffered medium and a reduced capacity for glucose oxidation by the pentose phosphate pathway. The pH of proliferating cultures supplemented with either 14 mM NaHCO₃ or with a mixture of organic buffers (pK 7.4) was increased by 0.3 pH unit over that of the controls. The rate of glucose oxidation by the pentose phosphate pathway in confluent cultures supplemented with NaHCO₃ or organic buffer increased by 60% 24 h after pH correction. This was associated with an increase in glucose uptake from the medium. We conclude that pH elevation in confluent heart cell cultures stimulates both growth and the capacity for glucose oxidation by the pentose phosphate pathway. The data also provide further evidence for a relationship between activity of the pentose phosphate pathway and cell growth.     

Koelle's copper thiocholine method performed with a low-pH phosphate buffer,followed by osmification of the precipitate: revival of two abandaned procedures

Summary Using a glutaraldehyde-fixed mouse neuromuscular junction, a fine precipitate of copper thiocholine was obtained with Koelle's medium prepared by a mixture of phosphate buffer (pH 5.6–5.9) and copper glycine solution (strongly acidic). The final pHs of these incubation media were very low, being situated between 3.8 and 4.2, respectively. It is well known that phosphate buffer, at such a low pH value, has no buffering effect on the acetic acid of enzymatic hydrolysis. This probably caused a sharp drop of the pH value in the vicinity of the enzymatic site and allowed a fine localization of copper thiocholine, the precipitation of which is pH dependent. Furthermore, the osmification of copper thiocholine in the same phosphate buffer provided a finely localized electron dense product. The chemical nature of the osmified copper thiocholine is discussed.     

Endospore formation by <Emphasis Type="Italic">Streptomyces avermitilis</Emphasis> in submerged culture

The ability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycle on synthetic medium CP1 with glucose under phosphate limitation and (2) in autolysing cell suspensions of high density obtained by tenfold concentration in phosphate buffer (pH 7.2) with 0.2% CaCl₂ of stationary-phase cells grown in synthetic medium. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to retain the main species-specific features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in surface cultures.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 204–214.Original Russian Text Copyright © 2005 by Filippova, Gorbatyuk, Poglazova, Soina, Kuznetsov, El-Registan.     

Elaboration of the method of "quiescent cells" for the study of heliomycin-synthesizing system of Streptomyces olivocinereus

A resting cell procedure was developed for S. olivocinereus. Washed mycelium of S. olivocinereus produced heliomycin for a short incubation period of 1.5 hours in a nitrogen-free medium containing a buffer solution, salts, a source of carbon and an inhibitor of protein synthesis. With the developed procedure production of heliomycin in the system of resting cells was investigated. For this purpose mycelium collected during various phases of S. olivocinereus development in batch cultures was used. It was found that in the batch cultures the rate of heliomycin production by the 24th hour of the development was comparable with that of the antibiotic accumulation in the resting cell system. After that period it markedly decreased by the 48th hour. This deviation in the dynamics of heliomycin production in batch cultures and the resting cell system can serve as a basis for further studies on heliomycin biosynthesis control by the carbon source.     

Cohesive properties of axenically grown cells of the slime mould Dictyostelium discoideum

It has been found that Dictyostelium discoideum cells from the exponential growth phase of axenically grown cultures are cohesive, whereas those from stationary phase are not. These differences in cohesiveness are seen in phosphate buffer and in axenic medium. Stationary phase medium inhibits the aggregation of log phase cells; stationary phase cells inoculated into freshly prepared medium regain their cohesiveness. Stationary phase medium may contain an inhibitor of cell cohesion. pH differences between the two types of medium are not entirely responsible for loss of cohesiveness.     

Extracellular ATP and ADA-buffer enable chick embryo fibroblasts to grow in secondary cultures in protein-free, hormone-free, extracellular growth factor-free medium

The replacement of HEPES with ADA buffer and addition of ATP to the serum-free, protein-free Eagle's minimal essential medium (EMEM) caused stimulation of proliferation in sparse, secondary cultures of resting chick embryo fibroblasts. Better cell growth and highly reproducible results were obtained if the cells had been dispersed from primary cultures with EGTA and thereafter remained without any further contact with serum or other extracellular proteins than when trypsin was used. The frequent changes of culture medium caused no retardation of cell growth. The observed more than 12 fold increase in the cell number after stimulation of the cultures with ATP is interpreted in favour of the conceptions assuming a significant role of intracellular calcium and intracellular pH in regulation of cell proliferation.     

Iron phosphate precipitation in Murashige and Skoog media

Murashige and Skoog revised medium, a standard plant tissue culture medium, precipitated on keeping because iron was ineffectively chelated. Most of the precipitate formed after two days and analysis indicated that it was predominantly iron phosphate. It was found that, after precipitation, the supernatant medium had lost c. 45% of its original iron, 20% zinc and 13% phosphate. The following modifications prevented precipitation: lowering the pH to below 3.2, increasing to 3 the molar ratio of EDTA:Fe or preparing the stock solution of FeEDTA with inadequate heating. Most of these modifications had a deleterious effect on the growth and greening of Ocimum basilicum suspension cultures. However, when an increase in the molar ratio of EDTA:Fe was achieved by reducing the iron concentration to one-third, there was no inhibitory effect on growth or greening of these suspension cultures.     

The capacity of growing algal cells to affect the pH and the buffering properties of the medium

Supernatants from cultures of green high-temperature algae,Chlorella 7-11-05 and Stichococcus 6-17-35, were used to obtaintitration curves and to calculate buffer indexes (ß).It was generally observed that the peak of buffering activityin growing cultures shifted to the pH characteristic of thepK of the newly generated buffer(s). Depending on the experimentalconditions and the age of the culture, the buffering capacityat its peak increased up to 4–5 times of the value forthe original medium. Buffering capacity of algal cells was demonstratedfor both strains grown in the media originally buffered eitherwith phosphate or with Tris and for cultures of different agesincluding those of the young synchronized cells. The capacityof growing cells to drastically affect the pH and/or the bufferingcharacteristics of the medium indicates that the role of thebuffer originally employed in a particular medium may oftenextend for a relatively short period of time after which thebuffer system(s) produced by the cells play a more importantpart. (Received May 18, 1971; )     

Starvation survival of Salmonella enteritidis.

Washed cells of Salmonella enteritidis harvested from a defined medium during logarithmic growth were subjected to starvation in pH 7 phosphate buffer at 37 C. Viability was measured by slide cultures and plate counts. The survival of cell suspensions equivalent to 1 to 10 mg (dry wt)/ml was influenced by cryptic growth. The rate of cryptic growth, assessed by plate counts, increased with cell density and could not be alleviated by starvation with dialysis. Dialysis of the starving culture did retard the onset of cryptic growth but did not eliminate it, indicating that the major substrates for regrowth were relatively large cellular components. In phosphate buffer, 6.7 homologous heat-killed cells allowed for the doubling of one S. enteritidis cell. Cryptic growth was not observed when cells were starved on the surface of membrane filters or in suspensions equivalent to 20 mug (dry wt)/ml (105 cells/ml). Similar half-life survival times were calculated for both these populations, but the shape of their survival curves differed significantly. These differences were attributed to stress factors encountered during cell preparation and during starvation. The half-life survival time of S. enteritidis starved at 20 mug (dry wt)/ml was 140 h in phosphate buffer, 82 h in 3,6-endomethylene-1,2,3,-6-tetrahydrophthalic acid buffer, and 77 h in tris(hydroxymethyl)aminomethane buffer.     

Stabilization of the synthetic media for plant tissue and cell cultures

In standardMurashige-Skoog medium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na₂EDTA. up to 10% free Fe^II ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na₂EDTA (at the approximate molar ratio Fe^II: Na₂EDTA 1: 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8 – 6.0. We also attempted to increase the relatively low buffering capacity of Murashige-Skoog medium. The addition of sodium citrate (1.25 mmol 1^-1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the Murashige-Skoog medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ± 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelation or of the buffering components.     

Involvement of Trihydroxyconjugated Bile Salts in Cholesterol Assimilation by Bifidobacteria

To determine the conditions of cholesterol assimilation, various strains of Bifidobacterium species were cultured in the presence of cholesterol and bile salts. During culturing, Bifidobacterium breve ATCC 15700 assimilates cholesterol in the presence of oxgall at pH values lower than 6. This strain was selected to study the influence of conjugated (taurocholic acid) and deconjugated (cholic acid) bile salts on cholesterol assimilation. B. breve ATCC 15700 assimilated cholesterol (up to 51%) when cultures were undertaken in the presence of taurocholic acid, whereas less than 13% of the initial amount of cholesterol was measured in the cells in the presence of cholic acid. Cultured in the presence of six individual di- or trihydroxyconjugated bile salts, bifidobacteria strains assimilated cholesterol. This assimilation appeared to be more important in the presence of trihydroxyconjugated bile salts (tauro- and glycocholic acids). It is concluded that trihydroxyconjugated bile salts are involved in the assimilation of cholesterol by bifidobacteria. Received: 20 June 1996 / Accepted: 19 July 1996     

Optimization of date syrup as a novel medium for lovastatin production by <Emphasis Type="Italic">Aspergillus terreus</Emphasis> ATCC 20542 and analyzing assimilation kinetic of carbohydrates

Lovastatin is a statin drug, which lowers cholesterol level in blood due to inhibition of (S)-3-hydroxy-3-methylglutaryl-CoA reductase. Date syrup is a rich medium for microbial growth and metabolite production. The main carbohydrates present in the date syrup are glucose and fructose. In this study, date syrup was used as a complex and bioresource medium for lovastatin production by Aspergillus terreus in the submerged cultivation. Optimization of the date syrup medium in order to achieve the highest titers of lovastatin and biomass was carried out. Four factors were studied by response surface methodology including concentration of date syrup carbohydrates, yeast extract concentration, pH, and rotation speed of the shaker. Optimal conditions for these factors found were as follows: concentration of date syrup carbohydrates, 64 g/l; yeast extract concentration, 15 g/l; pH, 6.5; and agitation speed, 150 rpm. It gave lovastatin concentration of 105.6 mg/l. Next, batch cultures in the optimal conditions were performed in a 2.5-l working volume bioreactor and led to the lovastatin titer of 241.1 mg/l during 12 days. Aspergillus terreus showed diauxic growth in the optimized medium with a shift from glucose to fructose assimilation during the run. Glucose and fructose assimilation kinetic parameters revealed that more lovastatin is produced during glucose assimilation, while more biomass was formed during fructose assimilation.     

Enhancement of oleandrin production in suspension cultures of Nerium oleander by combined optimization of medium composition and substrate feeding

Abstract

Oleandrin has been identified as the most potent antitumor ingredient of the Mediterranean herb Nerium oleander L. A strategy for optimization of medium compositions and conditions was developed for enhanced oleandrin production in suspension cultures from leaf-origin explants of Nerium oleander. The cell suspension cultures were grown in various modifications of MS medium as a basal medium. The effects of different natural extracts, plant growth substances, carbon and nitrogen sources and phosphate on the growth and oleandrin accumulation were investigated as well as effect of light, pH, shaking speed and substrate feeding. The highest oleandrin yield was obtained when the nitrogen concentration was lowered to two-thirds and the phosphate concentration increased by two-thirds of that specified in the MS medium in the presence of 3% sucrose, coconut milk, indolebutyric acid and benzyladenine in concentrations of 1 and 2 mg l^?1, respectively. Lower pH and faster shaking speed favored oleandrin accumulation. Chemical feeding of progesterone and cholesterol boosted the oleandrin concentration to higher levels reaching 8.23±0.05 mg g^?1 dry weight. This was about 10-fold higher than that detected in field-grown plants using the same extraction and analytic conditions, and about 24-fold higher than that determined in control cultures with regular MS medium and without precursor feeding.     

Effect of disruption by sonication under different conditions on the activity of glucose dehydrogenase from resting spores of Bacillus subtilis

The activity of glucose dehydrogenase present in resting spores of Bacillus subtilis varied strikingly with the conditions for disrupting the spores by sonic treatment, namely, the time and strength of sonication, and the type and pH of the solution used for suspending the spores. When the resting spores were sonicated for 30 min at a current of 1.45 A in 100 mM phosphate buffer in the range of pH 6.0 to 6.6 or in deionized water, the enzyme activity of the former suspension was approximately 10 times higher than that of the latter suspension. However, the enzyme activity of the latter was markedly stimulated in the presence of sodium chloride. The glucose dehydrogenase from resting spores disrupted in 100 mM phosphate buffer (pH 6.6) was a salt-independent, active enzyme with a molecular weight of about 120,000, whereas the enzyme from resting spores disrupted in deionized water was a salt-dependent, inactive one with a molecular weight of about 55,000. A high concentration of dipicolinic acid strongly inhibited activation by a salt of inactive glucose dehydrogenase from resting spores in deionized water, suggesting one of its several important roles in vivo.     

Possibility of association and activation of glucose dehydrogenase during germination and outgrowth of Bacillus subtilis spores

Both a salt-dependent form and an active form of glucose dehydrogenase [EC 1.1.1.47] were isolated from germinated spores of Bacillus subtilis disrupted in deionized water and 100 mM phosphate buffer (pH 6.6), and most of the enzyme isolated from young vegetative cells was the active form regardless of the conditions for breakage by sonication. The molecular weight of the salt-dependent form of the enzyme was about 55,000 and that of the active form was about 120,000. From the above results and the results on the glucose dehydrogenase extracted from resting spores disrupted in deionized water and 100 mM phosphate buffer (pH 6.6) reported in a previous paper, we propose that glucose dehydrogenase is present in resting spores as a monomeric form and is activated with association in vivo during germination and outgrowth.