A rapid method for separation and assay of radiolabeled mucopolysaccharides from cell culture medium

A method is described for the separation and assay of radiolabeled glycosaminoglycuronans (GAGs) from cell culture medium. Following papain digestion and precipitation with cetylpyridinium chloride onto cellulose ester filter membrane, hyaluronic acid can be separated from sulfated GAGs by selective elution with HCl.     

Use of ristomycin diffused into the culture medium for separation of meningococci from nasopharyngeal mucus

The method for enhancing the growth of meningococci after the inoculation of the specimens of nasopharyngeal mucus is proposed. The method is based on the diffusion of ristomycin into agar from ristomycin-impregnated filter paper strips. During the study of 694 specimens of nasopharyngeal mucus the above-mentioned method allowed isolation of 108 meningococcal cultures, while in serum agar only 21 cultures were isolated. The effectiveness coefficient of this method was found to be 80.6%, its effectiveness index being 5.1. The method facilitated the isolation of meningococci and in some cases reduced the time of analysis by 1 day.     

Shear treatment of starter culture medium improves separation behavior of Streptococcus thermophilus cells

A central step in the production of starter cultures is the separation of the cells from the fermentation medium, which is usually achieved by disk centrifuges. In case of microorganisms which produce exopolysaccharides (e.g., various strains of lactic acid bacteria), the properties of the respective exopolysaccharides may interfere with this separation step. By using six strains of Streptococcus thermophilus the hypothesis was tested that a shear treatment of the fermented culture medium improves subsequent cell separation markedly. Depending on the type of exopolysaccharides (freely present in the medium, or as capsules around the cells) an energy input of up to 2.5 kJ/mL generated with an Ultra‐Turrax affected cell chain length of the strains and viscosity of fermentation medium differently. For bacteria producing capsular exopolysaccharides, space‐ and time‐resolved centrifugation experiments revealed an increase of sedimentation velocity after shear treatment. In general, viability of the microorganisms, detected by flow cytometry measurements and fermentation experiments, was not affected by the shearing procedure. The results therefore indicate that strain‐targeted shearing is helpful to improve the separability of cells from the fermented media.     

Glycosidases from the culture medium of Physarum polycephalum.

Eight exo-glycosidase activities were detected in the axenic culture medium of the myxomycete, Physarum polycephalum. The secretion of each enzyme examined followed the growth curve and continued during the stationary phase after the cessation of growth. Two or more forms of each enzyme were detected after electrophoretic separation. The beta-N-acetyl-D-hexosaminidase activity was readily separated into its two electrophoretic forms, X and Y, which were purified 145- and 306-fold respectively. These beta-N-acetyl-D-hexosaminidases had several similar characteristics. Evidence is presented that the major electrophoretic form of alpha-D-galactosidase is heterogeneous. The possible functions of extracellular glycosidases in teir occurrence and properties.     

产鼠李糖脂菌株种子培养条件和C、N源的优化

对自行筛选的3个可利用废弃油脂进行发酵生产鼠李糖脂的铜绿假单胞菌菌株进行评价,并进行了种子培养条件和摇瓶发酵部分条件的优化。种子培养优化实验表明,当培养基pH 6～8,培养温度为30 ℃时最利于菌体生长。菌株均具有一定的耐盐性,在5%的盐度下生长未受到明显抑制,因此在沿海地区采用盐水或海水发酵具有较广阔的应用前景。通过排油圈、表面张力、苯酚-H2SO4比色法比较了这3个菌株的表面活性剂表面活性的大小,以表现较好的Z41进行了摇瓶发酵条件的优化。单因素实验表明,发酵较优条件为发酵温度30 ℃,接种量5%。在此基础上,通过正交试验对Z41菌株发酵培养基中的C、N源进行了研究,实验结果表明,在考虑因素间交互作用和发酵成本的情况下,最佳C源为3%炸货油,最佳N源为3.5 g/L尿素。在此发酵条件下,糖脂产量较高13.024 g/L,且成本较低。     

Mild method of pre-concentration of Dunaliella salina from culture medium

The photo- and chemo-taxisms of Dunaliella salina were used to induce a cell concentration in a layer of fresh water pumped to the top of a saline culture through a circular diffuser. When two consecutive operations were imposed to a 2.2 m 2 raceway type algae pond, 90% of the biomass (at 0.7 dry cells/l) was recovered in 16% of the initial volume. The biomass, free from chemicals and/or mechanical damage, could be centrifuged to obtain a high quality biomass for human consumption. The remaining medium in the pond (containing the rest of the cells) could be re-innoculated and allow a new cycle of production.     

New nonsynthetic medium for Rhizobium culture production from wastes

A nonsynthetic medium was formulated for placement of mannitol fully by saccharified pea husk (Pisum sativum L.) and water hyacinth (Eichhoornia crassipes) with Trichoderma viride QM 9414 and molasses. Yeast extract was Partially replaced by proteolysed pea husk, water hyacinth, and mycelium of T. viride QM 9414 by boiling 4 hr with 5% (v/v) HCl. The rhizobial growth was equal in both standard yeast extract mannitol (YEM) and formulated nonsynthetic media. However, barring Rhizobium phaseoli (urid) E-6, the rhizobial counts in thenon-synthetic medium were higher then the counts in YEM medium. In the fermentor, rhizobial growth was also almost equal to YEM medium. These results indicated that costly ingredients like mannitol and yeast extract can be replaced by hydrolysates of pea husk, water hyacinth, mycelium of T. viride, and molasses.     

Peptidomic analysis of blastocyst culture medium and the effect of peptide derived from blastocyst culture medium on blastocyst formation and viability

High‐quality in vitro human embryo culture medium can improve the blastocyst formation rate and blastocyst quality and be beneficial for the clinical application of single blastocyst transfer. Mammalian embryos can secrete protein products into the surrounding medium. As a group of bioactive molecules and degraded proteins, peptides have been shown to participate in various biological processes. Using liquid chromatography‐tandem mass spectrometry, we performed comparative peptidomic analysis of human culture medium in blastocyst formation and nonblastocyst‐formation groups. A total of 201 differentially expressed peptides originating from 157 precursor proteins were identified. Among these, a peptide derived from HERC2 (peptide derived from blastocyst culture medium [PDBCM]) passed through the zona pellucida, was distributed on the perivitelline space, was absent in arrest embryos and highly expressed in high‐quality blastocysts compared with low‐quality blastocysts, and significantly promoted blastocyst formation in a concentration‐dependent manner. These results indicate that PDBCM may be a novel biomarker for predicting blastocyst formation and viability. The mechanism remains unclear and needs to be explored in the future.     

Optimization of culture medium for enhanced production of exopolysaccharide from Aureobasidium pullulans

Polysaccharides produced by microorganisms are utilized for a variety of purposes, including the use in cosmetics and as food additives. More recently, polysaccharides have been exploited by the medical and pharmaceutical industries, and those originated from many species of mushrooms have been especially useful in industrial applications; however, the production and synthesis of these compounds is costly and time consuming. In this study, we developed a method for low-cost production of exopolysaccharide (EPS) that effectively screens components and optimizes medium composition using statistical methods (Plackett-Burman and Box-Behnken design). As a result, we obtained the following optimized medium: sucrose 165.73 g/L, sodium nitrate 3.08 g/L, dipotassium phosphate 1.00 g/L, potassium chloride 0.50 g/L, magnesium sulfate 0.50 g/L, ferrous sulfate 0.01 g/L, and 0.71 g/L of Ashbya gossypii extract. The maximum production of about 29 g/L EPS was achieved in the optimized medium during 84 h batch fermentation.     

Volatile fatty acids accumulation and rhamnolipid generation in situ from waste activated sludge fermentation stimulated by external rhamnolipid addition

The solubilization and acidification of waste activated sludge (WAS) were apparently enhanced by external rhamnolipid (RL) addition. The maximum solute carbohydrate concentrations increased linearly from 48 ± 5 mg COD L⁻¹ in the un-pretreated WAS (blank) to 566 ± 19 mg COD L⁻¹, and protein increased from 1050 ± 8 to 3493 ± 16 mg COD L⁻¹ at RL dosage of 0.10 g g⁻¹ TSS. The highest VFAs concentration peaked at 3840 mg COD L⁻¹ at RL dosage of 0.04 g g⁻¹ TSS, which was 4.24-fold higher than the blank test. RL was generated in situ during WAS fermentation when external RL was added. It was detected that RL concentration was increased from initial 880 ± 92 mg L⁻¹ to 1312 ± 7 mg L⁻¹ at the end of 96 h with RL dosage of 0.04 g g⁻¹ TSS, which was increased to 1.49-fold. Meanwhile, methane production was notably reduced to a quite low level of 2.0 mL CH₄ g⁻¹ VSS, showing effective inhibition of methanogens by RL (58.8 mL CH₄ g⁻¹ VSS in the blank). In addition, the activity of hydrolytic enzymes (protease and α-glucosidase) was enhanced accordingly. VFAs accumulation and RL generation in situ demonstrated that the additional RL substantially performed enhanced biological effects for waste activated sludge fermentation.     

Arabinogalactan-proteins from the suspension culture medium and plasma membrane of rose cells

Arabinogalactan-proteins (AGPs) that bind to beta-glycosyl Yariv antigens have been purified from the culture medium and plasma membrane of "Paul's Scarlet" rose cells. Starting from culture medium or from plasma membrane vesicles prepared by aqueous two-phase partitioning, the purification procedure involved Yariv antigen-induced precipitation and subsequent chromatographic steps. Two fractions, AGP-(a) and AGP-(b), were obtained from the culture medium, and one AGP fraction was obtained from the plasma membrane. The glycosyl compositions of all three fractions were dominated by arabinosyl and galactosyl residues and included glucuronosyl and other minor residues. Methylation analysis showed that AGP-(a) and AGP-(b) were both highly branched 3,6-galactans with terminal arabinofuranosyl substituents. The amino acid compositions of all three AGPs were high in alanine, hydroxyproline, and serine and/or threonine. The amino-terminal sequence of AGP-(b) contained an alanine-hydroxyproline repeat. While sharing general structural similarity, the AGPs from the plasma membrane and the culture medium were distinguishable by composition and by size and charge, with the plasma membrane AGPs being larger and more negatively charged than the culture medium AGPs.     

Physical separation of hemopoietic stem cells from cells forming colonies in culture

Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.