Development and experimental study of a dried nutrient medium for the microbiological diagnosis of streptococcal infections

The comparative study of different protein bases has shown that the combined base containing animal blood hydrolysate (amino peptide) and acidic casein hydrolysate, moderately cleaved, in the proportion 1:1 is a good source of nitrogen and ensures the intensive growth of streptococci. As determined by the study of the physiological parameters and growth of streptococci, the presence of fodder yeast extract, glutamine, glucose and phosphates in media containing blood hydrolysate and casein hydrolysate has been found to render a stimulating effect on the growth and multiplication of these organisms. The data thus obtained have been used as the basis for developing the formula of a dried culture medium, capable of ensuring the growth of streptococci without blood or serum added and not inferior in its quality to Todd-Hewitt Broth manufactured by Oxoid Ltd. (Great Britain) and Difco Laboratories (USA). The physico-chemical and physiological characteristics of the proposed medium have been determined. The use of the new dried culture medium in medical practice will make it possible to improve the microbiological diagnosis of streptococcal infections.     

Optimization of casein-based semisynthetic medium for growing of toxigenic Corinebacterium diphtheriae in a fermenter

Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.     

Chickpea protein hydrolysate as a substitute for serum in cell culture

The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.     

Manipulation of division symmetry and developmental fate in cultures of potato microspores

The effect of media composition on microspore culture was investigated in one tetraploid and two diploid potatoes. The viability of microspores isolated from 4.5 to 5 mm buds was in the range of 33 to 52%. In media for anther culture, microspores showed no further development and lost viability within 2 days. In M1 medium containing mineral components, sucrose, uridine, cytidine, myo-inositol, glutamine and lactalbumin hydrolysate, 18 to 37% of microspores underwent mitosis within 14 days. Up to 95% of the divisions were symmetric and produced equal nuclei. Some symmetrically divided microspores eventually produced structures with 3 to 10 nuclei. The proportion of the total microspore population producing multinuclear structures reached 9% in diploid clones responsive to anther culture and 1 to 2% in recalcitrant cv. Borka. Symmetric mitoses in M1 medium were induced in the presence of glutamine and lactalbumin hydrolysate. Nucleosides and myo-inositol had no effect on microspore division. In the absence of all organic components except sucrose, most mitoses were asymmetric, formation of multinuclear structures was reduced and most pollen accumulated starch indicative of gametophytic fate. In complete M1 medium, starch accumulation was suppressed. Suppression also occurred in asymmetrically divided microspores, indicating a direct inhibition of pollen development independent of the mode of microspore division. This inhibitory effect of M1 medium might present a stress which triggers the induction of symmetric microspore division and subsequent formation of multinuclear structures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of plant protein hydrolysates for varicella virus production in Serum-Free Medium

Serum-free media containing no animal-derived components were assessed for their efficacy to produce live attenuated varicella virus. Serum-free medium containing an ultrafiltrate of soy protein acid hydrolysate and lipid resulted in a viral production yield comparable to media containing fetal bovine serum, indicating that varicella virus can be produced without the risk of contamination associated with the use of bovine serum. Revisions requested; Revisions requested 20 December 2004; Accepted 21 December 2004     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

Optimization of a culture medium for xylanase production by Bacillus sp. using statistical experimental designs

The concentrations of oat spelt xylan, casein hydrolysate and NH4Cl in the culture medium for production of xylanase from Bacillus sp. I-1018 were optimized by means of response surface methods. The path of steepest ascent was used to approach the optimal region of the medium composition. The optimum composition of the nutrient medium was then easily determined by using a central composite design and was found to be 3.16g/l of xylan, 1.94g/l casein hydrolysate, 0.8g/l of NH4Cl. The xylanase production was increased by 135% when the strain was grown in the optimized medium compared to initial medium.     

Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sits increased during the first 2 days in culture to a plateau that was maintained for the next 2-3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.     

Dry medium for isolating Clostridium perfringens

The possibility of using blood substitutes (hydrolysin, casein hydrolysate "tsolipk" and amino peptide) with expired shelf life as a culture medium for the isolation of Cl. perfringens has been studied. Dry culture medium based on these inedible products has been developed. To stimulate bacterial growth, fodder yeast extract has been used. The suppression of the growth of extraneous microflora is ensured by the use of antibiotics: polymyxin sulfate and mycerin sulfate. The recommended medium is not inferior in its quality (sensitivity, rapidity of growth, differentiating and inhibiting properties, etc.) to media based on meat and casein. The use of the newly developed medium is economically grounded and allows one to obtain standard results.     

Low-cost culture medium for the production of proteases by <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> SA and their potential use for the preparation of antioxidant protein hydrolysate from meat sausage by-products

The present study aims to maximize proteases production by Bacillus mojavensis SA strain and their use to produce bioactive protein hydrolysates from a meat by-product. The production of SA bacteria proteases was maximized using a culture medium based on wheat bran, which offer an advantage in minimizing the production cost and enhancing the enzyme activity by using agro-industrial wastes. The composition of media and cultural conditions for optimal proteases production by B. mojavensis SA strain were investigated. A successful and significant improvement of the alkaline proteases production (four folds) by the SA strain was achieved using the medium composed of (g/l): wheat bran, 50.0; KH₂PO₄, 0.5; K₂HPO₄, 0.5; CaCl₂, 2.0; pH 6.0, where the growth conditions were monitored at 37 °C with an agitation speed of 200 rpm. Interestingly, the enzyme preparation of B. mojavensis was applied for the preparation of protein hydrolysates from a meat by-product. Hydrolysis was carried out for 180 min at pH 12.0. The resulting hydrolysate displayed an important antioxidant activity as evaluated by the radical scavenging capacity, the reducing power, and the β-carotene bleaching inhibition. The present study showed the high proteases’ producing level by B. mojavensis SA strain in a low-cost fermentation medium (wheat bran) and their potential use in the production of bioactive protein hydrolysate from meat by-products.     

Secretion of cyclodextrin glucanotransferase in E. coli using Bacillus subtilis lipase signal peptide and optimization of culture medium

The cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132 was fused to the secretive lipase signal peptide of B. subtilis. This leads to an efficient secretion of the recombinant enzyme into the culture medium of E. coli as an active and soluble form contrasting with the native construction leading to a periplasmic production. In order to enhance the yield of CGTase production, an experimental design methodology was applied for the optimization of the culture composition. Hence, the media components were submitted to preliminary screening using a Plakett-Burman design. The concentrations of the major operating ones were then optimized to enhance the secretion of CGTase using response surface methodology. The findings revealed that concentrations of 0.5% potato starch, 3% yeast extract, 3% tryptone, 1.5% casein hydrolysate, 0.5% NaCl, 0.2% KH2PO4, and 0.02% MgSO4 were the optimal conditions for CGTase production. The experimental value (9.43 U/mL) obtained for CGTase activity was very close to the predicted value (9.27 U/mL).     

Influence of medium and cold pretreatment on androgenetic response in Lolium perenne L.

Summary Genotypes of Lolium perenne L. with different androgenetic responses were used to test effects of induction medium composition. The media tested were potato II (pII), 190-2, and modified Linsmaier and Skoog media, LS-1, LS-2, and LS-3. The effect of different gelling agents, activated charcoal in a double layer design, and casein hydrolysate were also studied. From 36,696 anthers, 25,906 embryo-like structures, 1,959 albino and 173 green plants were generated. Significant differences were found between media, genotypes and medium-genotype interactions studied. All three media commonly used, pII, 190-2, and LS-3, were equivalent in production of green plants. Cold pretreatment of the anthers (4°C) significantly increased the number of embryo-like structures, the number and proportion of albino plants produced, but not the production of green plants.Abbreviations ELS embryo-like structures - ALB albino plants - ANT anthers - GRP green plants - DH doubled haploid plants - AC activated charcoal - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog (1965) basal medium - MS Murashige and Skoog (1962) - PL plants - pII potato II induction medium - DL double layer     

Optimization of the medium for the yeast synthesis of biotin

In order to increase the yield of biotin produced by the culture Sporobolomyces pararoseus, the medium containing sucrose, asparagine, MgSO4 (NH4)2SO4, KH2PO4, vitamin complex and trace elements was optimized. With the aid of a fractional factor experiment (2(5-1)) and a complete factor experiment (2(4)), the proportion of constituents was chosen in such a way as to double biotin yield, i.e. to increase it to 55.25 micrograms/l. An enrichment of the medium with yeast autolysate, casein hydrolysate and peptone in the presence of adenine increased biotin yield to 105.7 micrograms/l and cell productivity from 6.1 to 8.0 micrograms/l dry biomass.     

Neutral Red Assay for Measurement of Quantitative Vero Cell Cytotoxicity

A neutral red assay involving Vero cells was used to quantitate the cytotoxic activity of verotoxins (VT) produced by Escherichia coli and to investigate changes in titer caused by altering the composition of the cell culture medium. Three variations on medium 199 were investigated: one involved supplementing the medium with 5% fetal bovine serum (FBS), a second was the use of serum-free (SF) medium that contained 5% bovine serum albumin and 5 μg of fibronectin per ml, and the third involved the use of 4% Ultroser G, a commercial serum replacement. The level of cytotoxicity varied markedly with the type of VT and with the medium that was used. For VT1, there was no difference in cytotoxicity between medium with 5% FBS and SF medium, but cytotoxicity was reduced more than 100-fold in medium containing Ultroser G compared with cytotoxicity in the other media. For VT2, VT2v, and VTe, there was a slight reduction in cytotoxicity in medium containing 4% Ultroser G and a more marked reduction in SF medium compared with cytotoxicity in medium containing 5% FBS.     

Using highly efficient nonlinear experimental design methods for optimization of Lactococcus lactis fermentation in chemically defined media

Optimization of fermentation media and processes is a difficult task due to the potential for high dimensionality and nonlinearity. Here we develop and evaluate variations on two novel and highly efficient methods for experimental fermentation optimization. The first approach is based on using a truncated genetic algorithm with a developing neural network model to choose the best experiments to run. The second approach uses information theory, along with Bayesian regularized neural network models, for experiment selection. To evaluate these methods experimentally, we used them to develop a new chemically defined medium for Lactococcus lactis IL1403, along with an optimal temperature and initial pH, to achieve maximum cell growth. The media consisted of 19 defined components or groups of components. The optimization results show that the maximum cell growth from the optimal process of each novel method is generally comparable to or higher than that achieved using a traditional statistical experimental design method, but these optima are reached in about half of the experiments (73–94 vs. 161, depending on the variants of methods). The optimal chemically defined media developed in this work are rich media that can support high cell density growth 3.5–4 times higher than the best reported synthetic medium and 72% higher than a commonly used complex medium (M17) at optimization scale. The best chemically defined medium found using the method was evaluated and compared with other defined or complex media at flask‐ and fermentor‐scales. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Simultaneous determination of 33 amino acids and dipeptides in spent cell culture media by gas chromatography-flame ionization detection following liquid and solid phase extraction

A rapid, sensitive and reproducible gas chromatographic method with flame ionization detection is described for the simultaneous identification and quantification of 33 amino acids and dipeptides in spent cell culture media in under seven minutes. The method involves the use of the EZ:faast(Phenomenex) amino acid sample testing kit. Instrumental and assay precision, percent recovery, linear range, limit of detection and peak identity in highly complex cell culture media containing either soy hydrolysate or fetal bovine serum were validated using gas chromatography-flame ionization detector (GC-FID).     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from Brewer's spent grain hemicellulosic hydrolysate

Brewer's spent grain, the main byproduct of breweries, was hydrolyzed with dilute sulfuric acid to produce a hemicellulosic hydrolysate (containing xylose as the main sugar). The obtained hydrolysate was used as cultivation medium by Candidaguilliermondii yeast in the raw form (containing 20 g/L xylose) and after concentration (85 g/L xylose), and the kinetic behavior of the yeast during xylitol production was evaluated in both media. Assays in semisynthetic media were also performed to compare the yeast performance in media without toxic compounds. According to the results, the kinetic behavior of the yeast cultivated in raw hydrolysate was as effective as in semisynthetic medium containing 20 g/L xylose. However, in concentrated hydrolysate medium, the xylitol production efficiency was 30.6% and 42.6% lower than in raw hydrolysate and semisynthetic medium containing 85 g/L xylose, respectively. In other words, the xylose-to-xylitol bioconversion from hydrolysate medium was strongly affected when the initial xylose concentration was increased; however, similar behavior did not occur from semisynthetic media. The lowest efficiency of xylitol production from concentrated hydrolysate can be attributed to the high concentration of toxic compounds present in this medium, resulting from the hydrolysate concentration process.     

解淀粉芽胞杆菌PC2产抑菌物质培养基及发酵条件优化

【目的】优化解淀粉芽胞杆菌PC2产抑菌活性物质发酵培养基及发酵条件。【方法】以马铃薯葡萄糖液体培养基为基础,依据发酵液对金黄色葡萄球菌抑菌圈的单因素试验结果,采用Box-Behnken响应面法优化发酵培养基,二次通用旋转组合设计,频率分析法优化发酵条件。【结果】影响发酵液抑菌活性的培养基主要组分为马铃薯、蔗糖和L-谷氨酸钠,最优发酵培养基配方为:马铃薯188.0 g/L,蔗糖22.0 g/L,L-谷氨酸钠1.80 g/L,培养基成本为0.81元/L;最佳发酵条件为:接种量6%、发酵温度30°C、装液量40 mL/250 mL、摇床转速185 r/min、发酵时间24 h、初始pH 7.0。优化后发酵液对金黄色葡萄球菌抑菌圈直径为30.82 mm,较优化前的18.22 mm增加了12.60 mm。【结论】优化后的培养基和发酵条件提高了解淀粉芽胞杆菌PC2发酵液的抑菌活性,为该菌株的工业化生产应用提供了依据。     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.     

Improving probiotic spore yield using rice straw hydrolysate

Spore-forming Bacillus sp. has been extensively studied for their probiotic properties. In this study, an acid-treated rice straw hydrolysate was used as carbon source to produce the spores of Bacillus coagulans. The results showed that this hydrolysate significantly improved the spore yield compared with other carbon sources such as glucose. Three significant medium components including rice straw hydrolysate, MnSO₄ and yeast extract were screened by Plackett–Burman design. These significant variables were further optimized by response surface methodology (RSM). The optimal values of the medium components were rice straw hydolysate of 27% (v/v), MnSO₄ of 0·78 g l⁻¹ and yeast extract of 1·2 g l⁻¹. The optimized medium and RSM model for spore production were validated in a 5 l bioreactor. Overall, this sporulation medium containing acid-treated rice straw hydrolysate has a potential to be used in the production of B. coagulans spores.