Concentrated Cultures of Leuconostoc citrovorum

Two single-strain cultures of Leuconostoc citrovorum were grown in a broth medium with automatic pH control. Culture concentrates were prepared by centrifugally harvesting the cells and resuspending them in 1/50th the original volume in 10% nonfat milk solids. The concentrates were stored in liquid nitrogen until analyzed. The maximum population attainable was approximately equal when cultures were grown at pH 6.0, 6.5, or 7.0 with sodium hydroxide or ammonium hydroxide as the neutralizer. Citrate was required in the growth medium for the cultures to be able to produce diacetyl subsequently in milk. At pH 6.0, the cells reached maximum population and ability to produce diacetyl. Organoleptic analysis by an experienced flavor panel showed a preference for cottage cheese creamed with a creaming mixture prepared with a culture concentrate rather than a normal culture. The culture concentrates maintained their viability and ability to produce diacetyl for at least 30 days when stored in liquid nitrogen.     

Effect of Fermentation Conditions on Growth of Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091 in Pure and Mixed Cultures

Two strains of mesophilic lactic acid bacteria, Streptococcus cremoris AM2 and Leuconostoc lactis CNRZ 1091, were grown in pure and mixed cultures in the presence or absence of citrate (15 mM) and at controlled (pH 6.5) or uncontrolled pH. Microbial cell densities at the end of growth, maximum growth rates, the pH decrease of the medium resulting from growth, and the corresponding acidification rates were determined to establish comparisons. The control of pH in pure cultures had no effect on L. lactis CNRZ 1091 populations. The final populations of S. cremoris AM2, however, were at least five times higher than when the pH was not controlled (4 × 10⁸ vs. 2 × 10⁹ CFU · ml⁻¹). The pH had no effect on the growth rate of either strain. That of S. cremoris AM2 (0.8 h⁻¹) was about twice that of L. lactis CNRZ 1091. When the pH fell below 5, the growth of both strains decreased or stopped altogether. Citrate had no effect on S. cremoris AM2, while final populations of L. lactis CNRZ 1091 were two to three times higher (3 × 10⁸ CFU · ml⁻¹); it had no effect on the maximum growth rates of the two strains. Citrate attenuated the pH decrease of the medium and reduced the maximum acidification rate of the culture by 50%, due to the growth of S. cremoris AM2. Acidification due to L. lactis CNRZ 1091, however, was very slight. Regardless of the conditions of pH and citrate, the total bacterial population in mixed culture was lower (by 39%) than that of the sum of each pure culture. Mixed culture improved the maximum growth rate of L. lactis CNRZ 1091 (0.6 h⁻¹) by 50%, while that of S. cremoris AM2 was unaffected. The acidification rate of the growth medium in mixed culture, affected by the presence of citrate, resulted from the development and activity of S. cremoris AM2.     

Detection of Specific Strains and Variants of Streptococcus cremoris in Mixed Cultures by Immunofluorescence

Antisera against four different strains of Streptococcus cremoris were raised by injecting rabbits with washed suspensions of whole cells. These antisera interacted specifically with the corresponding strain in a mixture of up to nine different S. cremoris strains. The antisera could be used for analyzing the composition of mixed cultures containing these strains by immunofluorescence. Competition experiments were performed in batch and continuous cultures under amino acid limitation. A bacteriophage-sensitive variant of S. cremoris SK11 (SK1128) could be distinguished from a bacteriophage-resistant variant (SK1143) by the same immunofluorescence technique. The competition between the two variants and the stability of both variants in pure cultures were followed with the specific antibodies. Antibodies against the purified proteolytic system of S. cremoris Wg2 were used to determine the presence of proteases by immunofluorescence in several S. cremoris strains under different culture conditions. The described immunofluorescence methods can be used to analyze complex mixed starter cultures common in the dairy industry as the strains and variants present in these mixtures can be recognized microscopically.     

Some pink yeasts associated with softening of olives

Pink yeasts identified as Rhodotorula glutinis var. glutinis, R. minuta var. minuta, and R. rubra produce polygalacturonases which cause a slow softening of olive tissue. Both pectin methyl esterase and polygalacturonase are produced when cultures are grown in appropriate media. Crude, cell-free dialyzed enzyme preparations measured viscosimetrically exhibited optimal activity on sodium polygalacturonate at pH 6.0 and 40 C, and were active in the range of pH 4.0 to 9.0 and 10 to 50 C. Cultures grown in sterilized olives and brine at pH 4.0 with sterile glucose added aseptically caused a slow softening of tissue as measured with a Christel texturometer. Similar results were obtained when crude, cell-free enzyme preparations were added to olives in buffer solution at pH 6.0 with Merthiolate. Commercial control of these yeasts is easy if anaerobic conditions can be provided. Otherwise, the industry has to resort to manual removal of the film from the brine surface, either by skimming or by flagellation.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Selection of Protease-Positive and Protease-Negative Variants of Streptococcus cremoris

Protease-negative variants were shown to outcompete the wild-type strains of Streptococcus cremoris E₈, HP, and Wg₂ at pH values higher than 6.0 in milk. For S. cremoris E₈ this process was studied in more detail. At lower pH values the wild type had a selective advantage. This pH-dependent selection was not found in all media tested. The poor growth of the protease-negative variant at low pH was not due to lower internal pH values. By growing S. cremoris E₈ and Wg₂ in acidified milk (pH 5.9) the proteolytic activity of the cultures could be stabilized. In continuous cultures under amino acid limitation the wild type S. cremoris E₈ and HP strains had a selective advantage over the protease-negative variants at low dilution rates (D < 0.2) at all pH values of the medium. This was apparently due to a lower affinity-constant (K_s) of the protease-positive variants for amino acids. Finally, a high fraction of protease-positive variants could be maintained in continuous cultures by using a growth medium with low concentrations of casein as a nitrogen source. At high dilution rates nearly all cells were protease positive.     

Associative growth studies in three-strain mixtures of lactic streptococci

A recently developed differential agar medium was used to study associative growth patterns in 17 different heterologous, three-strain mixtures of Streptococcus lactis, S. cremoris, and S. diacetilactis grown in milk. Mixtures were made by combining equal volumes of 18-hr milk cultures of the three species. Relative populations of component species were followed through three successive transfers in milk after the initial mixed propagation. Direct evidence for strain dominance and compatibility was obtained. A procedure also was developed to estimate the extent of suppression of S. lactis and S. diacetilactis in a mixture containing a dominant S. cremoris strain. The technique described could be successfully applied in quality-control work in the dairy-starter manufacturing industry.     

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.     

Catenaria anguillulae in the mermithid nematode Romanomermis culicivorax

Disease epizootics in laboratory cultures of the mermithid nematode Romanomermis culicivorax were caused by a chytridiomycetous fungal parasite, Catenaria anguillulae. Techniques used for mass rearing the nematode provided ideal conditions for dispersal of zoospores and multiplication of the fungus; up to 90% of the postparasites of R. culicivorax were parasitized. The fungus was isolated and grown on yeast extract-soluble starch agar, and the effects of different temperature and pH regimes on the fungus were investigated as possible methods for prevention or control. A temperature suitable for mass rearing the nematodes but unsuitable for the fungus could not be found, as C. anguillulae had a broad temperature range. Adjustment of pH or a short period of chilling (0°C) were more promising control measures. At acid pH (<5), zoospore motility and germination and infection of postparasites were suppressed. The disease was controlled by rearing nematodes in water adjusted to a pH of 4.5.     

Effect of Lactose Concentration on the Efficiency of Plating of Bacteriophages on Streptococcus cremoris

The efficiency of plating of phages derived by ultraviolet induction of, or by lytic growth on, certain strains of Streptococcus cremoris was found to vary by as much as 10⁵ depending on the lactose concentration of the medium in which the indicator bacteria were grown and the length of time the stationary-phase indicator cultures were aged. This effect was noted only when the culture was used as an indicator for phages that had previously grown on an apparently unrelated strain of bacteria. Conditions of culturing and aging had no detectable effect upon the ability of a strain to serve as an indicator for phage that had previously been cultured on the same strain. These observations suggest the presence of some kind of physiologically labile restriction system in strains of S. cremoris. The implications of this finding for increasing the sensitivity of the host range test in determining phage susceptibility, whether from induced lysates, whey, or lytic phage stocks, are discussed. It is recommended that, for all such testing, the concentration of lactose in buffered media be increased to such levels as required to obtain a final pH similar to that of a freshly coagulated milk culture, namely, below pH 5.0.     

Nitrogenase activity and respiration of cultures of Rhizobium spp. with special reference to concentration of dissolved oxygen

Studies of nitrogenase in culture of the cowpea rhizobia (Rhizobium spp.) strains 32H1 and CB756 are reported. Preliminary experiments extablished that, even agar cultures were grown in air, suspension of bacteria prepared anaerobically from them were most active at low concentrations of free dissolved O₂. Consequently, assay for activity usd low concentrations of O₂, stabilized by adding the nodule pigment leghaemoglobin.In continous, glutamine-limited cultures of 32H1, nitrogenase activity appeared only when the concentration of dissolved O₂ in the cultures approached 1 μM. Lowering the glutamine concentration in the medium supplied to the cultured from 2 to 1 mM halved the cell yield and nitrogenase activity was also deminished. Omitting succinate from the medium caused the concentration of dissolved O₂ to rise and nitrogenase activity was lost. Upon restoration of the succinate supply, The O₂ concentration immediately fell and nitrogenase was restored. The activity doubled in about 8 h, whereas the doubling time of this culture was 14 h. Sonic extracts of 32H1 cells from continous cultures with active nitrogenase contained components reacting with antiserum against nitrogenase Mo-Fe protein from soybean bacteroids. Continous cultures grown at higher O₂ concentration, with only a trace of active nitrogenase, contained less of these antigens and they were not detected in highly aerobic cultures. Nitrogenase activity of a continous cultures was repressed by NH₄⁺; the apparent half-life was about 90 min.Cells of 32H1 from a continous culture growing at between 30 and 100 μM dissolved O₂ possessed a protective mechanism which permitted respiration to increase following exposure to a rapid increase in O₂ concentration from low levels (O₂ shock). This effect disappeared as disappeared as the O₂ concentration for growth was reduced towards 1 μM.     

Indentification of huperzine A-producing endophytic fungi isolated from Huperzia serrata

This present study was designed to investigate the production of huperzine A (HupA), an acetylcholine inhibitor, which was produced by an endophytic fungi isolated from Huperzia serrata. Screening of 94 endophytic fungal isolates obtained from plant H. serrata was carried out for the production of HupA. Their morphological characteristics were studied and rDNA sequence analysis was carried out. The cultures were grown in liquid culture medium and the extracted metabolites were analyzed by thin layer chromatography and high performance liquid chromatograph for the presence of HupA. The DPPH scavenging ratio and inhibition ratio of acetylcholinesterase (AchE) of the same were determined. 3 out of 94 strains i.e. S29, L44 and S94 showed significant AchE-inhibitory activity and antioxidant activity. Strain L44 which exhibited maximum yield of HupA (37.63 μg/g on dry weight basis) was identified as Trichoderma species by ITS sequence analysis. In conclusion, endophytic fungi from H. serrata can be used as a new resource of HupA.     

Two Uptake Systems for Fructose in Lactococcus lactis subsp. cremoris FD1 Produce Glycolytic and Gluconeogenic Fructose Phosphates and Induce Oscillations in Growth and Lactic Acid Formation

Fructose transport in lactococci is mediated by two phosphotransferase systems (PTS). The constitutive mannose PTS has a broad specificity and may be used for uptake of fructose with a fructose saturation constant (K_Fru) of 0.89 mM, giving intracellular fructose 6-phosphate. The inducible fructose PTS has a very small saturation constant (K_Fru, <17 μM), and the fructose 1-phosphate produced enters the Embden-Meyerhof-Parnas (EMP) pathway as fructose 1,6-diphosphate. Growth in batch cultures of Lactococcus lactis subsp. cremoris FD1 in a yeast extract medium with fructose as the only sugar is poor both with respect to specific growth rate and biomass yield, whereas the specific lactic acid production rate is higher than those in similar fermentations on other sugars metabolized via the EMP pathway, e.g., glucose. In fructose-limited chemostat cultures, the biomass concentration exhibits a strong correlation with the dilution rate, and starting a continuous culture at the end of a batch fermentation leads to large and persistent oscillations in the biomass concentration and specific lactic acid production rate. Two proposed mechanisms underlying this strange growth pattern follow. (i) Fructose transported via the fructose PTS cannot be converted into essential biomass precursors (glucose 6-phosphate or fructose 6-phosphate), because L. lactis subsp. cremoris FD1 is devoid of fructose 1,6-diphosphatase activity. (ii) The fructose PTS apparently produces a metabolite (presumably fructose 1-phosphate) which exerts catabolite repression of both mannose PTS and lactose PTS. Since the repressed mannose PTS and lactose PTS are shown to have identical maximum molar transport rates, the results indicate that it is the general PTS proteins which are repressed.     

Metabolism of aromatic acids by Polyporus hispidus

When grown on glucose as principal carbon source the culture medium of Polyporus hispidus was found to contain phenolic acids, including p-coumaric and caffeic acids. ¹⁴C-Studies indicated that phenylalanine is converted to cinnamic acid as well as to phenylpyruvic acid and tyrosine in cultures. Cell-free preparations of mycelium contained phenylalanine and tyrosine ammonia-lyse activities and were capable of effecting the hydroxylation of cinnamic, p-coumaric and benzoic acids.     

Conjugal Transfer of Bacteriophage Resistance Determinants on pTR2030 into Streptococcus cremoris Strains

Agar surface conjugal matings were used to introduce heat-sensitive phage resistance (Hsp⁺) determinants carried on the conjugal plasmid pTR2030 into Streptococcus cremoris KH, HP, 924, and TDM1. Lactose-fermenting (Lac⁺) transconjugants were selected from matings of Lac⁻ variants of S. cremoris KH, HP, 924, and TDM1 with Streptococcus lactis ME2 or a high-frequency donor, S. lactis T-EK1 (pTR1040, Lac⁺; pTR2030, Hsp⁺). For all of the S. cremoris strains examined, select Lac⁺ transconjugants were completely resistant to plaquing by their homologous lytic phages. In all cases the plaquing efficiencies were less than 10⁻⁹. Acquisition of a 30-megadalton plasmid (pTR2030) in the S. cremoris phage-resistant transconjugants was demonstrated by direct plasmid analysis, by hybridization with ³²P-labeled probes, or by conjugal transfer of pTR2030 out of the phage-resistant transconjugants into a plasmid-cured recipient, S. lactis LM2302. Acid production, coagulation ability, and proteolytic activity of phage-resistant transconjugants in milk were comparable to those of their phage-sensitive parents. Further, S. cremoris phage-resistant transconjugants were not attacked by phage in starter culture activity tests, which included a 40°C incubation period. The results demonstrated that phage resistance determinants on pTR2030 could be conjugally transferred to a variety of S. cremoris strains and confer resistance to phage under conditions encountered during cheese manufacture. Phage-resistant transconjugants of S. cremoris M43 and HP were also constructed without the use of antiblotic markers to select conjugal recipients from mating mixtures.     

Effects of culture medium and auxins on growth of adventitious root cultures of Cuphea aequipetala Cav. and their ability to produce antioxidant compounds

Cuphea aequipetala Cav. (Lythraceae), a species highly valued for its medicinal properties, is threatened in the wild. To provide an alternative source of material for production of bioactive compounds, we established adventitious root cultures of C. aequipetala and determined their phenolic compounds contents and antioxidant activity. Cultures were initiated from root tips of in vitro C. aequipetala plantlets and were grown in B5 or SH culture medium containing either indole butyric acid (IBA) or α-naphthalene acetic acid at 0, 5 or 10 µM. The maximum root biomass (1.6 g/L dry mass (DM) per L medium) was recorded after 14 days of growth in B5 + 5 µM IBA. Roots in B5 medium remained green, whereas they tended to oxidize in SH medium. The highest contents of total phenolic compounds (9.1 ± 0.1 µg gallic acid equivalents/g DM) and flavonoids (37.5 ± 0.7 µg quercetin equivalents/g DM) were in roots grown in B5 + 5 µM IBA after 14 days of growth. Root cultures accumulated mainly flavan-3-ols, whereas roots or leaves from whole plants accumulated mainly flavonols. We analyzed the antioxidant properties of root extracts using in vitro assays. Roots grown in B5 medium showed stronger free-radical scavenging activity than that of roots grown in SH medium. Our results show that adventitious root cultures of C. aequipetala are a promising system for research on antioxidant compounds biosynthesis and for scaled-up production of useful biological materials.     

Glutathione Protects Lactococcus lactis against Oxidative Stress

Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to ~60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H₂O₂ compared to the resistance of cells without intracellular glutathione. The resistance to H₂O₂ treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H₂O₂. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.     

Purification and Some Properties of Diplococcin from Streptococcus cremoris 346

Eleven of 150 Streptococcus cremoris strains examined produced the bacteriocin diplococcin. The diplococcin activity spectrum was restricted to S. cremoris and Streptococcus lactis strains, and none of a wide range of other gram-positive or gram-negative strains were inhibited. The diplococcin produced by S. cremoris 346 was purified by ammonium sulfate precipitation and column chromatography. Purified diplococcin was very unstable at room temperature and lost 75% of its activity after heating at 100°C for 1 min. The proteolytic enzymes trypsin, pronase, and α-chymotrypsin completely inactivated diplococcin. The amino acid composition showed a high content of acidic and neutral acids and a correspondingly low content of basic amino acids, including one residue of ornithine per mole. From the amino acid analysis a molecular weight of 5,300 was estimated. Diplococcin was readily distinguished from the S. lactis bacteriocin nisin by its restricted activity spectrum, its biological properties, and by cross-reaction experiments.     

Control of N-acetylglucosaminidase specific activity in myxamoebae of Dictyostelium discoideum

Myxamoebae of Dictyostelium discoideum were grown with Aerobacter aerogenes as substrate, on nutrient agar plates or in shaken culture, with mean doubling times that varied between 3 and 6 hr. Growth in axenic culture was with mean doubling times of 5–6 or 8 hr. The specific activity of N-acetylglucosaminidase was three to four times higher in myxamoebae grown axenically than in myxamoebae grown with A. aerogenes and there was no correlation between enzyme specific activity and myxamoebal growth rate. High specific activities of N-acetylglucosaminidase were also found in myxamoebae grown with gram-positive bacteria (Bacillus megaterium, Staphylococcus lactis) as substrate while low specific activities were found in myxamoebae grown with the gram-negative bacteria Acinetobacter lwoffi and Pseudomonas aeruginosa as well as with A. aerogenes. A preparation of the cell envelope of A. aerogenes was nearly as effective as the intact bacteria at depressing myxamoebal N-acetylglucosaminidase specific activity. Lipopolysaccharide extracted from the cell envelope of gram-negative bacteria, or the lipid A component of the lipopolysaccharide, also depressed N-acetylglucosaminidase specific activity when added to axenic cultures of myxamoebae.     

Agar medium for differential enumeration of lactic streptococci

An agar medium containing arginine and calcium citrate as specific substrates, diffusible (K₂HPO₄) and undiffusible (CaCO₃) buffer systems, and bromocresol purple as the pH indicator was developed to differentiate among lactic streptococci in pure and mixed cultures. Milk was added as the sole source of carbohydrate (lactose) and to provide growth-stimulating factors. Production of acid from lactose caused developing bacterial colonies to seem yellow. Subsequent arginine utilization by Streptococcus lactis and S. diacetilactis liberated ammonia, resulting in a localized pH shift back toward neutrality and a return of the original purple indicator hue. The effects of production of acid from lactose and ammonia were fixed around individual colonies by the buffering capacity of CaCO₃. After 36 hr at 32 C in a candle oats jar, colonies of S. cremoris were yellow, whereas colonies of S. lactis and S. diacetilactis were white. S. diacetilactis, on further incubation, utilized suspended calcium citrate, and, after 6 days, the citrate-degrading colonies exhibited clear zoning against a turbid background, making them easily distinguishable from the colonies of the other two species. The medium proved suitable for quantitative differential enumeration when compared with another widely used general agar medium for lactic streptococci.