Bacillus subtilis RBT-7/32 and Bacillus licheniformis RBT-11/17 as New Promising Strains for Use in Probiotic Feed Additives.

The normal functioning of a gastrointestinal microflora in poultry and livestock is of significant importance, since its imbalance negatively influences an organism's functions. In this study, the UV mutagenesis and selection were used to obtain two Bacillus strains possessing antagonistic activity towards Escherichia coli and Staphylococcus aureus, and their potential as a probiotic feed additive was evaluated. Compared to the parental strains, the ability of B. subtilis RBT-7/32 and B. licheniformis RBT-11/17 strains to suppress E. coli increased by 77 and 63%, respectively; the corresponding ability of these strains to suppress S. aureus increased by 80 and 79%, respectively. RBT-11/17 could not utilize microcrystalline cellulose and carboxymethyl cellulose, whereas cellulolytic activity of RBT-7/32 was doubled compared to the initial strain. The amylolytic activity of new strains was increased by 40%. Cultivation of strains on media containing soybean, pea, and corn meal did not provide any difference in the biomass production compared to the control. The heating of a water suspension of a dried biomass of the strains for 10-20 min at 80 and 100 °C or incubation in water solutions of citric, ascorbic, acetic, and formic acids (pH 3.0) for 3 and 24 h at 40 °C did not provide any negative influence on the spore survivability. Both strains were evaluated for their resistance to a number of veterinary antibiotics. Thus, RBT-7/32 and RBT-11/17 strains have good prospects for use in feed additives.

Biotransformation of Androstenedione by Filamentous Fungi Isolated from Cultural Heritage Sites in the State Tretyakov Gallery.

The transformation of steroids by microorganisms is widely used in medical biotechnology. A huge group of filamentous fungi is one of the most promising taxa for screening new biocatalytic reactions in order to obtain pharmaceutically significant steroids. In this work, we screened 10 filamentous fungi-destructors of egg tempera for the ability to biotransform androst-4-en-3,17-dione (AD) during cultivation in a liquid nutrient medium or in a buffer solution. These taxonomically unrelated strains, belonging to the classes Eurotiomycetes, Dothideomycetes and Sordariomycetes, are dominant representatives of the microbiome from halls where works of tempera painting are stored in the State Tretyakov Gallery (STG, Moscow, Russia). Since the binder of tempera paints, egg yolk, contains about 2% cholesterol, these degrading fungi appear to be a promising group for screening for steroid converting activity. It turned out that all the studied fungi-destructors are able to transform AD. Some strains showed transformation efficiency close to the industrial strain Curvularia lunata RNCIM F-981. In total, 33 steroids formed during the transformation of AD were characterized, for 19 of them the structure was established by gas chromatography/mass spectrometry analysis. In this work, we have shown for the first time that fungi-destructors of tempera paintings can efficiently transform steroids.

New bacitracin-resistant nisin-producing strain of Lactococcus lactis and its physiological characterization.

Nisin A belonging to the class I bacteriocins and produced by Lactococcus lactis subsp. lactis is widely used in many countries as highly efficient and safe preservative preventing growth of undesirable bacteria in food products. Though this compound is efficient at very low concentrations, reduction of its manufacturing cost is still relevant problem. An increased nisin A production requires improved resistance of its producer to nisin. According to some studies, mechanisms of microbial resistance to nisin A and bacitracin have a similar basis, and the same transporters are used to export these antibiotics from cells. To obtain strains with improved growth rate and nisin A productivity, selection of spontaneous bacitracin-resistant L. lactis mutants followed by examination of their stability as well as physiological and fermentation characteristics was carried out. Spontaneous mutants were obtained by culturing of L. lactis VKPM B-2092 strain on selective bacitracin-containing agar medium. The obtained bacitracin-resistant strain FL-75 was characterized by accelerated growth rate, doubled biomass accumulation, and improved nisin A resistance. The nisin A productivity of FL-75 exceeded that of the parental strain by 25% reaching 8902 U/mL after 14-h cultivation. In addition, FL-75 was characterized by the improved resistance to oxidative stress that has never been reported earlier for bacitracin-resistant microorganisms. Based on the performed characterization of FL-75, we can consider it as a new independent strain promising for the industrial production of food and feed biopreservatives. Comparison of published data and the obtained results allowed us to suppose that the bacitracin resistance mutation in FL-75 is determined rather by an increased expression of a gene homologous to the bcrC gene of Bacillus sp. than by the activation of multidrug resistance mechanisms. The revealed resistance of FL-75 to bacitracin and oxidative stress can be regulated by a common transcription factor activating in response to various environmental stresses.

Scaling up a virginiamycin production by a high-yield Streptomyces virginiae VKM Ac-2738D strain using adsorbing resin addition and fed-batch fermentation under controlled conditions.

Virginiamycin produced by Streptomyces virginiae as a natural mix of macrocyclic peptidolactones M and S is widely used in the industrial production of ethanol fuel and as an antibiotic feed additive for cattle and poultry. Its main antimicrobial components, M1 and S1 factors, act synergistically if the M1:S1 ratio in the final product is 70-75:25-30. This fact significantly complicates the development of stable high-yield strains suitable for industrial application. In the previous work, authors obtained a mutant S. virginiae VKM Ac-2738D strain, characterized by a high productivity in flasks and the optimum M1:S1 ratio (75:25) in the final product. In this study, the scale-up of the virginiamycin production by VKM AC-2738D from shake flasks to a pilot-scale (100 L) stirred fermentor was carried out and the possibility of the in situ use of synthetic adsorbing resins to remove virginiamycin from culture broth was assessed. After the optimization of pH and dissolved oxygen concentration (6.8-7.0 and 50%, respectively), the fed-batch fermentation of VKM Ac-2738D with continuous addition of 50% sucrose solution (5 g/L/day starting from 48 h of fermentation) resulted in a final virginiamycin titer of 4.9 g/L. Among four tested resins, Diaion® HP21 added to fermentation medium prior to sterilization absorbed 98.5% of the total virginiamycin that simplifies its further recovery procedure and increased its total titer to 5.6 g/L at the M1:S1 ratio of 74:26. The developed technology has several important advantages, which include (1) the optimum M1:S1 ratio in the final product, (2) the possibility to use sucrose as a carbon source instead of traditionally used and more expensive glucose or D-maltose, and (3) selective binding of up to 98.5% of produced virginiamycin on the adsorbing resin.

Strain Improvement of Streptomyces xanthochromogenes RIA 1098 for Enhanced Pravastatin Production at High Compactin Concentrations.

Pravastatin is one of the most popular cholesterol-lowering drugs. Its industrial production represents a two-stage process including the microbial production of compactin and its further biocatalytic conversion to pravastatin. To increase a conversion rate, a higher compactin content in fermentation medium should be used; however, high compactin concentrations inhibit microbial growth. Therefore, the improvement of the compactin resistance of a producer still remains a relevant problem. A multi-step random UV mutagenesis of a Streptomyces xanthochromogenes strain RIA 1098 and the further selection of high-yield compactin-resistant mutants have resulted in a highly productive compactin-resistant strain S 33-1. After the fermentation medium improvement, the maximum bioconversion rate of this strain has reached 91 % at the daily compactin dose equal to 1 g/L and still remained high (83 %) even at the doubled dose (2 g/L). A 1-year study of the mutant strain stability has proved a stable inheritance of its characteristics that provides this strain to be very promising for the pravastatin-producing industry.

Complete mitochondrial genome of compactin-producing fungus Penicillium solitum and comparative analysis of Trichocomaceae mitochondrial genomes.

We determined the complete mitochondrial genome sequence of the compactin-producing fungus Penicillium solitum strain 20-01. The 28 601-base pair circular-mapping DNA molecule encodes a characteristic set of mitochondrial proteins and RNA genes and is intron-free. All 46 protein- and RNA-encoding genes are located on one strand and apparently transcribed in one direction. Comparative analysis of this mtDNA and previously sequenced but unannotated mitochondrial genomes of several medically and industrially important species of the Aspergillus/Penicillium group revealed their extensive similarity in terms of size, gene content and sequence, which is also reflected in the almost perfect conservation of mitochondrial gene order in Penicillium and Aspergillus. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirmed the monophyletic origin of Eurotiomycetes.