Nataliya V. Karpova

Оптимизация питательной среды для глубинного культивирования штамма Mycolicibacterium neoaurum AС-3067D – продуцента β-каротина

Пробиотическая кормовая добавка с микробным бета-каротином: разработка и свойства

The biotechnological production of carotenoids offers a promising alternative to their chemical synthesis or extraction from plants. Mycolicibacterium species have shown potential as pigment-producing microorganisms. However, bacterial strains typically exhibit lower productivity compared to fungal and yeast strains. Earlier, we enhanced the β-carotene biosynthesis in M. neoaurum strain VKM Ac-3067D by modifying the cultivation medium. Key changes included replacing glucose with glycerol and soybean meal with skimmed milk powder (SMP) and increasing the urea content from 0.5 to 1.0 g/L. To further optimize β-carotene yield, a steepest ascent method was applied combining factorial design with a gradient-based optimization (Wilson–Box method). The resulting regression model showed that the most influential factors were the glycerol concentration and SPM use. The in-flask fermentation of the Ac-3067D strain in a medium containing 25.5 g/L of glycerol (carbon source) and 12.80 g/L of SMP (nitrogen source) increased β-carotene yield to 318.4 ± 8.3 mg/kg. In a 15 L bioreactor, β-carotene yield increased to 432.3 ± 10.4 mg/kg, while the biomass concentration reached 23.2 ± 1.2 g/L. The further scaling up to a 100 L bioreactor increased both β-carotene yield (450.4 ± 8.2 mg/kg) and biomass concentration (25.2 ± 1.1 g/L). Thus, β-carotene production technology using the M. neoaurum strain AC-3067D was successfully scaled up from 750 mL flasks to a 100 L bioreactor, confirming its potential for industrial-scale application.

The effect of carotene-containing probiotic feed additive on the digestibility and assimilation of feed and zootechnical indices of growing piglets was studied. The study involved 40 crossbred male piglets (F1: Duroc‒Landrace) with a live weight of 20‒25 kg. Two groups were formed, each with 20 piglets, based on the principle of analogues. The experiment was performed during a fattening period and lasted 30 days. The experimental group received a complete feed supplemented with the studied feed additive that resulted in a 6.8% increase in the daily weight gain compared to the control group. Piglets from the experimental group showed increased digestibility coefficients for dry matter (+2.2%), organic matter (+2.5%), and protein (+2.1%) compared to the control group. Additionally, the experimental group demonstrated a trend towards increase in the protein fractions in blood serum as well as the same trend for the phagocytic activity, phagocytic index, and phagocytic count. The study also demonstrated a significant reduction in the number of Salmonella and Enterococcus cells in the experimental group. The obtained results allow us to recommend this feed additive for use in the diet of fattened piglets.

Carotenoids represent a group of isoprenoid pigments whose high biological activity is not limited to their provitamin properties. Due to their ability to participate in redox reactions, carotenes are increasingly considered as promising compounds in the prevention and correction of cardiovascular and neurodegenerative disorders, as well as in oncology and the treatment of various other diseases. Carotenoids are widely used in the manufacture of food additives and dyes, feed for aquaculture, farm animals and poultry, as well as in so-called nutraceuticals and cosmetics. When formulating optimal feeding rations, vitamin A nutrition is often considered separately due to its vital role in normal growth, development, maintenance and reproduction. The main precursor of vitamin A is β-carotene, which naturally enters the body exclusively via vegetable-based provender. However, since the carotene contained in plant raw materials is an unstable compound, the use of feed additives containing β-carotene becomes relevant. In industry, carotenoids can be produced either by chemical or biological synthesis. However, the majority of carotenoids – 80–90% – are obtained by chemical synthesis. At the same time, public demand for sustainable production dictates the need to find alternative approaches for obtaining this valuable commodity. The article provides an overview of the main biotechnological methods for the production of carotenes using various microorganisms, including microalgae, bacteria and fungi, as well as analysing the effect of culture conditions on the yield of target pigments.

A continuing interest of scientists regarding the development of new β-carotene production technologies is due to the high biological activity of this compound and its wide application range. Bacteria are considered among the possible β-carotene producers convenient for industrial use. The purpose of this study was to develop a Mycolicibacterium neoaurum strain with an enhanced ability for β-carotene production and to optimize the fermentation conditions to improve the final yield of the target compound. Using chemical mutagenesis with N-nitroso-N-methylurea along with further strain selection, a M. neoaurum strain Ac-501/22, whose productivity was 2.7-fold higher than that of the parental strain Ac-501, was developed. The effect of nitrogen and carbon sources as well as cultivation conditions on the growth of M. neoaurum Ac-501/22 and β-carotene production were studied to select the optimal fermentation regime. Due to an increase in the temperature of cultivation from 30 to 35 °C, replacement of glucose with glycerin (20.0 g/L) and degreased soybean flour with powdered milk (10.0 g/L), and increase in the urea content from 0.5 to 1.0 g/L, the level of β-carotene production was improved to 183.0 mg/kg that was 35% higher than in the control. Further strain fermentation in a 3 L bioreactor using an optimized medium with the pH level maintained at 7.0–7.2 and 50% pO₂ provided the maximum output of the target compound (262.4 mg/kg of dry biomass) that confirmed the prospects of the developed strain as an industrial β-carotene producer.

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.

Botrytis cinerea, causing grey mold, is a dangerous plant pathogen able to infect agricultural crops during the whole production cycle, including storage and transportation. A wide set of pathogenicity factors, high ecological plasticity, and universality of propagation and spreading of this fungus significantly complicate the control of this pathogen. A rapid increase in pathogen tolerance to fungicides dictates the necessity of developing antiresistant protection strategies, which include the use of biopreparations based on antagonistic microorganisms or their metabolites. The purpose of the study was to evaluate the antifungal activity of a dry biomass of P. chrysogenum VKM F-4876D (DMP), both individually and in combination with tebuconazole-, fludioxonil-, or difenoconazole-containing compounds recommended to control grey mold, in relation to B. cinerea isolated from grape samples. A water suspension of DMP was added to the PDA medium at a concentration of 1.0, 2.5, 5.0, 7.5, and 10.0 g/L. The pathogen growth inhibition was evaluated after 3, 7, and 14 days of cultivation; fungal cultures grown on DMP-free medium were used as a control. The resulting effective DMP concentration was 2.5 g/L. The effective concentrations of fungicides included in the study were determined to be 0.5 mg/L (tebuconazole), 0.1 g/L (difenoconazole), and 0.04 mg/L (fludioxonil). Combining DMP (2.5 g/L) with tebuconazole, difenoconazole, or fludioxonil (all taken at the effective concentrations) resulted in pathogen growth inhibition after 7 days of incubation by 86.5, 85.6, and 84.6%, respectively. Among all studied variants, the DMP (2.5 g/L) + difenoconazole (1.0 mg/L) combination provided the most efficient control of B. cinerea development under in vitro conditions: even after 14 days of incubation, the pathogen growth suppression remained at the level of 51.3%, whereas the DMP combination with tebuconazole or fludioxonil provided only 28.5 and 37.4%, respectively. The obtained results show good prospects for the efficient control of grey mold development, together with the reduction of pesticide loads on agrobiocoenoses and the prevention of the emergence of new resistant forms of plant pathogens.

The biological control of fungal crop diseases based on the use of micro-organisms or their metabolites is a promising environmentally friendly alternative to common fungicide-based technologies. Penicillium fungi which synthesize various biologically active compounds with a wide range of antimicrobial activity are of special interest as potential producers of antifungal biopreparations. The purpose of this study was the evaluation of the antifungal activity of a dry biomass of the Penicillium chrysogenum VKPM F-4876D strain (DMP), both individually and combined with commercial azoxystrobin-, fludioxonil-, difenoconazole-, and tebuconazole-based fungicides commonly used to control early blight, towards Alternaria solani, an infectious agent of this potato disease. The study was performed under in vitro (cultivation on solid and liquid media) and in vivo (detached potato leaves) conditions. The cultivation of A. solani on agarized medium supplemented with 5 g/L of DMP caused significant morphological changes in pathogen colonies, whereas 7.5 g/L of DMP resulted in complete suppression of pathogen development. DMP addition to the liquid culture of A. solani significantly increased electrolyte release, i.e., impaired the cell membrane integrity in the pathogen. Combining DMP (1–2 g/L) with fungicides at a dose range of 0.01–10 mg/L resulted in significant in vitro growth inhibition of A. solani. The best result was obtained for the combination of DMP (1 g/L) with fludioxonil (1–5 mg/L) or tebuconazole (10 mg/L): a significant and prolonged antifungal effect (96–97% growth inhibition) was observed during the whole 14-day period of observation. The treatment of detached potato leaves with 1 g/L of DMP prior to artificial infection with A. solani resulted in a four-fold increase in the number of uninfected leaves and a two-fold reduction in the number of severely infected leaves compared to the control. These results demonstrate the possibility to develop an efficient environmentally friendly approach to manage potato early blight and provide prerequisites for the reduction in pesticide load on agrobiocoenoses, thus contributing to the restoration of ecological balance and the prevention of the emergence and spread of resistant pathogen strains.

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.