Cultivation of Neisseria meningitidis serogroups A, B and C in a synthetic nutrient broth

The processes of the cultivation of N. meningitidis, serogroups A, B and C, in a liquid synthetic culture medium have been studied. Strictly group-specific biomass has been obtained. The maximum productivity at all stages of the batch cultivation of N. meningitidis strains 125 and 133 in this medium does not differ from that at similar stages of cultivation in modified Cohen-Wheller semisynthetic medium. In the serotype antigen preparations obtained from N. meningitidis strain 125 grown in the above-mentioned liquid synthetic culture medium basic polypeptides with a molecular weight of 33000, 36000 and 41000 D have been detected. Their presence in N. meningitidis cells is linked with the growth phase of the population.     

Nutrient requirements of Bacillus anthracis strains and the growth characteristics of strain STI-1 in batch cultivation on synthetic broths

A synthetic culture medium is proposed. This medium satisfies the nutritional requirements of most cultivated strains under study by a single amino acid: valine. The possibility of detecting strains with other nutritional requirements has been shown. The determination of LD50 in white mice has revealed no differences between the virulence of valine-dependent strains and that of strains with other nutritional requirements. During the batch cultivation of strain STI-1 in a liquid medium glucose shows the effect of substrate limitation and inhibition. The optimum concentration of glucose can be different, depending on the composition of the medium. The influence of different amino acids, the sources of carbon and energy, and nitrogen on the growth of the strain is evaluated.     

Dried nutrient media for the cultivation of pneumococcus and meningococcus

Dried synthetic nutrient medium for the cultivation of meningococci and the accumulation of their biomass has been developed. The kinetics of the culture growth, changes in pH and in the content of dissolved oxygen in the medium during prolonged controlled processes of the cultivation of meningococci in a bioreactor with the use of this medium have been studied. The stable physico-chemical properties and composition of the polysaccharide-protein complex isolated from the biomass of meningococci grown in the above-mentioned medium have made it possible to use it for the preparation of the samples of group B meningococcal vaccine. In addition, dried semi-synthetic nutrient medium for the accumulation of pneumococci without the necessity of introducing blood or serum into the medium has been developed. As regards its biological properties, this newly developed medium make it not inferior to meat media, containing blood or serum, and ensures good yield of biomass.     

Trial of different nutrient media for the production of an intracellular thermolabile enterotoxin by Escherichia coli strains H74-114 and 86

The conditions of cultivation, ensuring the maximum accumulation of intracellular thermolabile enterotoxin in the cultures of two E. coli strains of different origin, have been studied. Culture media manufactured in the USSR have been selected and the conditions of cultivation, necessary for obtaining intracellular thermolabile enterotoxin in preparative amounts, have been established. Under these conditions the yield of thermolabile enterotoxin in 1.4 mg per 1 liter of culture medium for strain H74-114 and 1.0 mg per 1 liter of culture medium for strain 86.     

Erratum to: Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Evaluation of a modified culture medium for Borrelia burgdorferi sensu lato

The aim of the present study was to assess the possible use of a modified medium, prepared in the laboratory using the constituents of Barbour-Stonner-Kelly (BSK) medium and medium 199 as base, for the culture of Borrelia strains, comparing the growth of individual strains in this medium and in the BSK-H medium, and the protein profile and antigenic characteristics of Borrelia proteins expressed in these media. A qualitative evaluation of growth of Borrelia species was made with acceptable results (morphology and motility), but during a quantitative evaluation using the three main genospecies of Borrelia, the better results were obtained with a B. burgdorferi sensu stricto strain. The modified medium did not enable the growth of a B. afzelii strain. The protein profile and antigenic characteristic of the expressed proteins in the modified medium were studied with satisfactory results. These results suggest the modified medium as an alternative for the cultivation of Borrelia strains, with some limitations, in poorly-resourced laboratories.     

Development of growth medium from meat processing waste products and assessment of its properties

New growth medium based on inedible substrate - meat processing waste products of slaughterhouses - was developed. New medium was characterized by physical, chemical, and biological properties using exacting and non-exacting microorganisms, as well as by periodical cultivation of Corynebacterium diphtheriae strain and obtaining the preparation of its antigens. The experimental medium provided the growth of chosen test-strains with typical properties. From biomass obtained during the periodic cultivation of model toxigenic strain of C. diphtheriae on liquid experimental growth medium, preparation with antigenic properties was extracted. It has been shown that biologic characteristics of experimental growth medium did not differ from those of meat-peptone medium that allows to use it for cultivation of bacteria from various taxonomic groups.     

Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Kinetics of growth and development of Mycobacterium rubrum under conditions of periodic culturing

The kinetics of growth, development, carotenogenesis and changes in the composition of the reaction medium were studied in the course of Mycobacterium rubrum batch cultivation in synthetic and complex media. The culture growth was found to be composed of five phases in the synthetic medium and of four phases in the complex medium. When the culture was grown in the synthetic medium, the phenomenon of distinct diauxy was recorded by changes in the specific growth rate as well as the economic and metabolic coefficients. These growth changes were shown to be associated with the fact that the culture started to assimilate sucrose instead of glucose at the subsequent growth phases, and carbotenogenesis was found to be uncoupled with the period of active biomass and protein synthesis.     

基于响应面分析的高抗原活性猪水肿病大肠杆菌疫苗培养基的研制及效果评价

【背景】猪水肿病大肠杆菌引发的疾病造成了很大的危害,但现有培养基存在培养密度低的问题。【目的】研制出高抗原活性猪水肿病大肠杆菌疫苗培养基。【方法】以常用的市售猪水肿培养基为对照,通过单因素试验、爬坡试验(Plackett-Burman, PB)、响应面(Box-Behnken, BB)试验对猪水肿培养基进行响应面优化,得到猪水肿培养基最优配方。以响应面试验得到的培养基培养猪水肿病大肠杆菌,评价不同培养时间点菌株的抗原活性,制作灭活疫苗,进行动物免疫保护试验。【结果】对研制的培养基进行扩大培养验证,发现扩大培养得到的菌株活菌数可达5×10⁹ CFU/mL以上,约为对照组的2倍。制备的灭活疫苗效价可达1:140 000,并在9 h时抗原蛋白效价达到最高。【结论】本研究研制出的疫苗培养基显著提高了猪大肠杆菌菌体密度,并可提高菌体抗原活性,为猪水肿病灭活疫苗的制备提供了技术指引。     

Cultivation of Leptospira in a liquid culture medium with lysed rabbit blood

A new method of the preparation of liquid culture medium for the cultivation of Leptospira has been developed on the basis of multiple freezing and thawing of defibrinated rabbit blood. A higher productivity of the new medium in comparison with the medium used in common practice has been established. The medium having a new composition is suitable for cultivation of Leptospira and the accumulation of biomass. The advantage of the medium made is in addition to high growth properties, the its economic efficiency and availability for practical laboratories.     

High cell density culture of Yarrowia lipolytica using a one-step feeding process

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by Lactobacillus bifermentans

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, or incubation period) and culture medium composition (mineral salts, carbon and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. On the other hand, the strain required for growth Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g. ammonium citrate). The bacterium had no requirement for sodium acetate for both growth and production of isomerases. The production rate of enzymes was increased when metal ions were added and mainly manganese (2.5 mM).     

Features of lipogenesis of basidiomycetes Pleurotus ostreatus and Flammulina velutipes in cultures on various media

The compositions of free fatty acids (FA) in the mycelia of oyster cap (Pleurotus ostreatus (Jacq. ex Fr.) Kumm.) and honey mushroom (Flammulina velutipes (Curt. ex Fr.) Sing.) and the effect of mycelium cultivation conditions on the composition and proportions of individual FA were investigated. Palmitic and linoleic acids were found to be major acids produced by P. ostreatus growing on solid agar medium and in a submerged culture with a synthetic medium. The composition of minor FA in P. ostreatus was dependent on cultivation conditions. Surface cultivation of its mycelium yielded pentadecanoic, octadecenoic, and stearic acids. Submerged cultivation additionally yielded undecanoic, myristic, hexadecenoic, and lignoceric acids. The composition of free FA in F. velutipes showed no significant differences from that of P. ostreatus. Variation in the C/N ratio in the cultivation medium affected both the FA composition in P. ostreatus and F. velutipes and the relationship between saturated and unsaturated FA.     

Minimal synthetic nutrient medium for Clostridium stricklandii bacteria

A composition of minimal culture medium for the anaerobic bacterium Clostridium sticklandii strain CSG was determined. A fully synthetic culture medium promoting the cell yield up to 1 g dry biomass per 1 1 was found. It is composed of 13 amino acids, sodium formiate, four vitamins, microelements and salts. The strain under study does not utilize glucose as a carbon and energy source.     

Antibiotic properties of Lactobacillus acidophilus, "Narine" strain,and the ways for their improvement

L. acidophilus strain 317/402 "Nariné" shows high antimicrobial activity with respect to pathogenic microorganisms. The degree of the activity of lactobacilli depends on cultivation conditions and the composition of the culture medium and its acidity, age of the culture, conditions of technological process, etc. The antimicrobial inhibition concentration of the product "Nariné", ensuring in vitro activity against Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio NAG, etc., has been established.     

Establishment of cell strains from human urothelial carcinoma and their morphological characterization

We have examined the conditions for cultivation of enzymatically dispersed cells from 34 human urothelial transitional cell carcinomas (TCC) of various types. By employing two culture methods, stationary and tapping suspension, and by using the synthetic medium DM 160 supplement with human umbilical cord serum and fetal bovine serum, six cell strains were established. In two strains the tapping suspension culture method was suitable for growth of highly malignant cancer cells that detach easily from the glass surface in stationary cultures. Each of the six cell strains has been maintained in culture for over 30 months with repeated subcultures of 32 to 128 times. The histopathological features of the original TCC were three differentiated papillary types and three anaplastic nonpapillary types. In two cell strains from TCC with low malignancy, however, the cancer masses that formed in nude mice differed from the original TCC in which they became more malignant, and one cell strain resembled the original TCC closely. In three stationary culture cell strains the epithelial nature was demonstrated by the presence of desmosomes and tonofilaments. In one cell strain only tonofilaments were present. In two tapping suspension culture cell strains the presence of desmosomes was not shown clearly, but fine tonofilaments were observed in one cell strain.     

Xylanase of the micromycete Rhizopus var. microsporus 595: preparation, structural and functional characteristics, and application

Procedures for the production of endo-1,4-beta-xylanase have been developed. An active producer-Rhizopus var. microsporus BKMF-595-has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to ensure maximum yield of the target enzyme. Activity of xylomicrosporin Px equaled 123 U/g, while the activity of xylomicrosporin Gx equaled 25 U/cm3. Homogeneous enzyme preparations, purified 59.44-fold and 72.6-fold, have been obtained. The dependence of endo-1,4-beta-xylanase catalytic activity on temperature and pH of the reaction medium has been studied. The enzyme has been shown to be most stable in the pH range 5.0-6.0 and to be thermostable. Amino acid composition and subunit structure of the enzyme were determined; the molecular masses of the subunits equaled 50 and 56 kDa. Carboxyl groups of glutamic and aspartic acid residues of the active center were experimentally shown to play an important role in catalysis. The potential of this enzyme for beer production has been demonstrated.     

Optimization of culture medium and growth conditions for production of L-arabinose isomerase and D-xylose isomerase by <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis>

Lactobacillus bifermentans was used to produce the intracellular enzymes L-arabinose isomerase and D-xylose isomerase. Various factors of cultivation (temperature, pH, and incubation period) and culture medium composition (mineral salts, carbon source, and nitrogen source) were studied to select the conditions that maximize production of these enzymes. Arabinose isomerase and xylose isomerase activities were 9.4 and 7.24 U/ml, respectively. They were highest at 9 h of cultivation in the optimized medium, 1.6 times higher than that in the basic MRS broth. The optimal medium composition and cultivation conditions were determined. For optimal growth, the strain required Tween 80 (1 g/l) and a source of inorganic nitrogen (e.g., ammonium citrate). The bacterium had no requirement for sodium acetate for either growth or production of isomerases. The production rate of enzymes was increased when metal ions were added, primarily manganese (2.5 mM). The text was submitted by the authors in English.     

Directed biosynthesis of proteolytic enzymes in Actinomyces thermovulgaris]

The biosynthesis of proteolytic enzymes in the thermophilic culture of Actinomyces thermovulgaris, strain T-54, was directed by changing the composition of the medium and the temperature of cultivation. A temperature of 40 degrees C is optimal for the growth and production of neutral and alkaline proteases. The maximum activity of acid proteases was found during the growth on a complex medium at 30 degrees C. An increase of temperature to 50 degrees C during the cultivation of the microorganism on a chemically defined medium resulted in its secondary growth and a sharp rise in the activity of alkaline and neutral proteases.