Development of the technology for preparation of enzymatic hydrolysate of waste chick embryos

The development of technologies for preparation of protein nutritional bases for microbiological nutrient media, from production waste of mainly readily available or non-food products, is a promising area in biotechnology. Researchers of Vyatka State Agrotechnological University assume that non-food secondary raw materials, such as waste chick embryos (WCEs) used in the production of anti-influenza products, could be used for these purposes, after removal of the virus-containing allantoic fluid. The aim of the study was to develop a technology for preparation of WCE enzymatic hydrolysate (WCEEH), and to evaluate growth properties of the hydrolysate-based solid nutrient medium, using Escherichia coli M-17 and Pseudomonas alcaligenes IP-1 test strains. Materials and methods: the authors offer methodological approaches to obtaining WCEEH and substantiate hydrolysis parameters. The obtained WCEEH was characterised in terms of physico-chemical properties: pH, amine nitrogen, total nitrogen, sodium chloride, degree of protein cleavage. The growth properties of the hydrolysate-based nutrient medium were studied using E. coli M-17 and Ps. alcaligenes IP-1 test strains. Results: the experiments demonstrated the feasibility of performing enzymatic hydrolysis of WCEs, and assessed physico-chemical properties of the prepared WCEEH batches. The study demonstrated the possibility of using the prepared hydrolysate as a component of solid nutrient media for growing the selected test strains. Conclusions: the study substantiated the optimal technological parameters for WCE enzymatic hydrolysis: pH (7.6 ± 0.3), duration (48 ± 2 h), temperature (49 ± 1) °C. The loading of hydrolysis components was optimised: mass fraction of the substrate—500 g/L, mass fraction of the hydrolysing agent—100 g/L. The physico-chemical properties of WCEEH make it suitable for preparation of microbiological media; the hydrolysate-based solid nutrient medium consistently ensures the growth of E. coli M-17 and Ps. alcaligenes IP-1 test strains with standard properties. The growth properties of the experimental medium are comparable to those of the meat-peptone broth-based nutrient medium.

Assessment of the intensity of damage to sunflower caused by artificial infection with septoria in phytotron conditions

The aim of our study was to analyze the development of Septoria leaf spot in sunflower seedlings artificially infected with the mycelium suspension of the pathogen in the phytotron and to determine the degree of damage caused by this disease in the lines of different origins. The materials for the experiment were self-pollinating sunflower lines ZL22A, ZL58A, ZL78A (all of Zaporizhzhya breeding the Institute of Oilseeds Crops of NAAS) and line HAR7 (originating from the USA). The research was conducted in the phytotron facility at the Department of Genetics and Plant Resources of Zaporizhia National University. The seeds of selected sunflower samples were sown into the soil in the specially prepared pots containing drainage and a mixture of chernozem and sand in equal proportions to a depth of 3 cm in two rows of 10 seeds each, with a distance between seeds of 0.5-1 cm. The plants grew and developed on a photoperiod of 16/8 hours (day / night) at a temperature of 23-25oC and a relative humidity of 65%. All samples were watered when needed and received equal amounts of water. Sunflower plants were inoculated in accordance with generally accepted methods of phytopathology. Next, the infected plants were placed in a humid chamber. The plants were infected with the inoculum of a 30-day old mycelium culture of the fungus Septoria helianthi, grown by cultivating the pathogen on a solid nutrient medium. The isolation of pathogen in pure culture as well as preparation of inoculum for infection were carried out using methods which were generally accepted in phytopathology and mycology, supplemented by author's own methodology. The degree of damage to the plants was assessed by visual examination of all leaves, using a 5-point scale, modified for our studies. The affected lines were determined by the percentage of plants with a certain degree of damage. The results of this research showed that sunflower lines sustained varying degrees of damage and, accordingly, varying degrees of resistance to Septoria. The least affected was line HAR7, with 70% of the plants in this line having damaged leaves. The most damaged of those were cotyledons and the first pair of true leaves, whereas just 11.7% of plants had the second pair of true leaves damaged. Completely unaffected plants made up 29.5% of this line. Line ZL22A was affected by 75%. Almost all plants in this line had damaged cotyledons and the first pair of leaves, with 70%. Besides, there were 5% of plants that had all their leaves affected. The number of unaffected plants was 25%. It should be noted that the ZL22A line was close in degree of damage to that of the HAR7 line and showed relative susceptibility to Septoria under phytotron conditions. ZL58A and ZL78A lines were the most affected by Septoria, with 100%. In the ZL58A line, there were 56.2% of plants with damaged cotyledons and the first pair of true leaves, and 43.8% of plants whose second pair of true leaves showed the symptoms of the disease. The plants of the ZL78A line with lesions on the cotyledons and the first pair of true leaves made up 60%, whereas the number of plants with the symptoms of Septoria leaf spot on the second pair amounted to 40%. In general, these two lines were characterized by the same degree of damage and can be considered susceptible to Septoria leaf blight. The disease progression on the HAR7 line is 38.2%, on the ZL22A line is 40%, on the ZL78A line is 60%, on the ZL58A line is 60,9%. The findings show that sunflower plant lines that were artificially infected with an aqueous suspension of fungal mycelium containing Septoria leaf spot pathogen in the conditions of phytotron developed the disease rather rapidly and exhibited varying degrees of damage. The proposed method provides a reliable infection of sunflower samples with the pathogen S. helianthi, a rapid assessment of the intensity of damage to sunflower lines by septoria.

PHOSPHATSOLUBILIZING BACTERIA OF THE GENUS PSEUDOMONAS AND THE EFFICIENCY OF THEIR APPLICATION TO INCREASE THE AVAILABILITY OF PHOSPHORUS

Phosphorus is the second most important element for plants after nitrogen. Fertilizers based on it, used to stimulate productivity, are inaccessible for most crops, which leads to their accumulation in the soil and environmental pollution. The use of phosphate-solubilizing bacteria increases the amount of phosphorus absorbed by plants. In most publications describing this group of bacteria, their effectiveness is assessed only in vitro by the halo zones formed on agar media with calcium orthophosphate. The aim of this study was to compare the solubilizing properties of bacteria of the genus Pseudomonas on a solid nutrient medium, as well as in sand and soil. It was shown that all studied cultures of microorganisms are capable of solubilizing insoluble phosphate in Pikovskaya's medium. The most active strains were Pseudomonas laurentiana ANT 56 and Pseudomonas sp. IB 182, isolated from the activated sludge of biological treatment facilities and arable soil, respectively. Experiments with the introduction of strains showed that the amount of mobile phosphorus in the sand increased 2.6-3.8 times in two weeks (in the control 1.2 times), while in the experiment with soil, a significant increase in the content of mobile phosphorus compared to the control was recorded only for the strain P . laurentiana ANT 17 (by 29.1%). It is assumed that the high solubilizing activity of the P. laurentiana ANT 17 strain may be due to the complex action of mechanisms of different nature, including the synthesis of indolyl-3-acetic acid and exopolysaccharide. The studies carried out make it possible to consider this bacterial strain as a promising object for creating on its basis a biological preparation for agricultural purposes.

The Effect of Long-Term Storage on Mycobacterium bovis

It was established that when stored for many years (10–13 years) in low-temperature conditions (3°C), without sub-culture on a nutrient medium, Mycobacterium bovis grew as visible colonies along the line of inoculation. However, due to long-term storage in conditions of low temperature (3°C) morphology of mycobacteria differed significantly from initial cultures formed by rod-shaped bacteria. Some of them became pigment-forming and smooth on the surface. Unlike the initial strain of mycobacteria, a perennial bacteria stored under hard conditions did not cause the death of guinea pigs or their sensitization to a purified protein derivative for mammals. Morphological forms of the perennial mycobacteria had the following changes: pigment forming, L-forms of the vesicular type, non-acid-fast thread-like (filamentous) bacillary forms, and elementary bodies when compared to the initial strain. There were also some genetic changes in the target DNA due to the long-term storage of M. bovis. It may indicate a mutation in the pathogen’s DNA. These mycobacteria had altered biochemical activity during storage. The number of passages on the solid nutrient medium did not affect their fermentative activity. However, the low cultivation temperature increases mycobacterial catalase activity and the ability to hydrolyze Tween-80.

Dissociated forms of moraxella isolated from the affected eyes of cattle

The dissociation phenomenon of epizootic cultures of Moraxella was studied. The study was conducted in economic entities of Almaty region of the Republic of Kazakhstan for 233 heads of cattle with clinical signs of keratoconjunctivitis. Isolation of the causative agent of Moraxella was performed by bacteriological washes from the conjunctival sacs of the eyes of animals. The laboratory study was carried out according to the approved methodological guidelines. It was found that bacteria of the genus Moraxella dissociate when grown on a solid nutrient medium for more than 6 hours in a thermostat at 37 °C. The bacteria were studied by the following methods: staining according to White-Wilson, thermoagglutination and acriflavine assay. When evaluating the grown colonies according to White-Wilson, the optimal dilution for crystal violet was found to be 1 : 2000, and for gentian violet stain 1 : 1000. In this case, the colonies in the S-form have a dark purple color with a metallic tint, and the dissociated colonies in the R-form do not stain. In the presence of dissociated cells, precipitation (thermoagglutination), sediment formation and clearing of the supernatant fluid at 90 °C for 30 minutes were noted. The suspension of undissociated colonies remained cloudy. When weighing microbial cells isolated by a bacterial loop from individual grown colonies in a solution of acriflavine, dissociated bacteria stick together to form conglomerates. When studying the antigenic activity of the S-, R- forms of Moraxella, it was revealed that the activity of the S-antigen significantly exceeded that of the R-forms. Data on the dissociation of Moraxella cultures can be used for the development of diagnostic and prophylactic drugs against moraxellosis in cattle.

BIOLOGICAL ACTIVITY OF N-{3-[(4-METHYLBENZENE-1- SULFONYL)IMINO]-6-OXOCYCLOHEXA-1,4-DIEN-1-YL}ARYLAMIDES AND THEIR DERIVATIVES

N-{3-[(4-Methylbenzene-1-sulfonyl)imino]-6-oxocyclohexa-1,4-dien-1-yl}arylamides and their derivatives were synthesized by the reaction of the corresponding N-(4-oxocyclohexa-2,5-dien-1-ylidene)arylsulfonamides with N-chloramides. The biological activity of the synthesized compounds was studied on test cultures of Escherichia coli 67, Staphylococcus aureus 209-p, Mycobacterium luteum VKM B-868 and fungi Candida tenuis VKM Y-70, Aspergillus niger VKM F-1119 by the method of diffusion of substances into agar on a solid nutrient medium. The degree of activity of the test compounds was determined by the diameter of the zones of inhibition of growth of test cultures of microorganisms. The minimum inhibitory, bactericidal and fungicidal concentrations were determined by the method of serial dilutions of the substance in a liquid nutrient medium. At the studied concentration, the method of diffusion of substances into agar on a solid nutrient medium has shown that these compounds have low activity against bacteria Escherichia coli, Staphylococcus aureus, Mycobacterium luteum and fungi Candida tenuis, Aspergillus niger. The diameters of the zones of inhibition of growth of test cultures of microorganisms were less than 15 mm. In research by the method of serial dilutions of the substance in a liquid nutrient medium, they have been found to have bactericidal and fungicidal activity. The minimum inhibitory concentration of N-{2-hydroxy-5-[(4-methylbenzene-1-sulfonyl)amino]phenyl}benzamide was 31.2 μg/ml against bacteria Mycobacterium luteum. The minimum inhibitory concentration of N-{2-hydroxy-3-methyl-5-[(4-methylbenzene-1-sulfonyl)amino]phenyl}benzamide against fungi Aspergillus niger was 31.2 μg/ml. The minimum bactericidal concentration of N-{2-hydroxy-5-[(4-methylbenzene-1-sulfonyl)amino]phenyl}benzamide was 62.5 μg/ml against bacteria Mycobacterium luteum. Minimum fungicidal concentration of N-{3-[(4-methylbenzene-1-sulfonyl)imino]-6-oxocyclohexa-1,4-dien-1-yl}benzamide and N-{2-hydroxy-3-methyl-5-[(4-methylbenzene-1-sulfonyl)amino]phenyl}benzamide was 62.5 μg/ml in action to mold fungi Aspergillus niger.

ASSESSMENT OF ANTIMYCOBACTERIAL ACTIVITY OF PROPYLTHIADIAZOLOQUINAZOLINE DERIVATIVES AGAINST MYCOBACTERIUM INTRACELLULARE

The compounds studied are propylthiadiazoloquinazoline derivatives, tryptanthrin analogs. Tryptantrin and its derivatives were found to have different antimycobacterial activity in vitro and in vivo. This series of experiments is devoted to the study of antimycobacterial activity of three propylthiadiazoloquinazoline derivatives obtained in the laboratory of organic synthesis and Biopharmaceutics of the Institute of Chemistry of the Academy of Sciences in Moldova using a non-tuberculous strain of Mycobacterium intracellulare, which is a part of the Mycobacterium avium complex (MAC). Species of the MAC are one of the main pathogen types causing mycobacteriosis. In this regard, propylthiadiazoloquinazoline derivatives are of interest for a comprehensive study as a potential antimycobacterial drug. To study antimycobacterial activity the method of dilutions in a dense nutrient medium of Middlebrook 7H9 broth with glycerol in Petri dishes was used. To assess antimycobacterial activity visual assessment of M. intracellulare growth in a solid nutrient medium was used. It is shown that the compounds studied inhibit M. Intracellulare growth. The most active compound was compound № 1 - 2-mercapto-5H-[1,3,4]-thiadiazolo-[2,3-b]-quinazoline-5-one.

Analysis of Antibiotic Resistance of Vibrio Сholerae Isolated From Environmental Objects in Russia in 2019

The aim of the study was to analyze the resistance to antibacterial drugs of Vibrio cholerae strains isolated from environmental water bodies on the territory of Russia in 2019.V.cholerae O1 El Tor (14) and V.cholerae nonO1/nonO139 strains were used in this work. Sensitivity/resistance to 11 antibacterial drugs was determined using the method of serial dilutions in a solid nutrient medium. The presence of drug resistance genes was determined using real-time PCR. Fluctuations in sensitivity/resistance of V.cholerae were found in various years. The phenotypic resistance of the strains to tetracycline and trimethoprim/sulfamethoxazole correlated with the presence of the tetR and dfrA1 genes in them. The presence of ICE was not detected in V.cholerae strains containing the tetR and qnrVC1 genes. The variability and wide spectrum of V.cholerae resistance require close attention to the problem of antibiotic resistance of cholera. The detection of ICE in the studied V.cholerae strains, as well as antibiotic resistance genes not associated with ICE elements, emphasize the need for molecular genetic monitoring of V.cholerae antibiotic resistance.

The use of plastic details to increase the contact surface of the substrate during the cultivation of attached species of algae

A series of experiments was carried out using strips (and nets) of synthetic polymers to increase the contact surface during the cultivation of attached forms of microphytes. We used bands of different composition, as well as with different microrelief surface areas, and a fine-mesh PLA network with an inhomogeneous surface. The experiments were performed on liquid nutrient media in accumulative cultures, and on solid nutrient medium in pure culture. The results showed a number of positive features of such cultivation, based on the selective isolation of individual species and their small combinations from the composition of accumulative cultures, as well as on the effective growth of individual species on specific elements of the microrelief of polymer surfaces. The polymers used were: PET, PP, LDPE, UHMWPE and PLA.

The resistance of chernozem soil microorganisms to soluble copper compounds

Аim. Determination of resistance of Ukrainian chernozem soil microorganisms to the influence of toxic copper(II). Methods. Content of copper-resistant microorganisms in chernozem was determined by counting colonies on a solid nutrient medium containing Cu(II). Resistance of microorganisms was determined by cultivation in the medium with concentration gradient of Сu2+. Results. Microorganisms resistant to toxic copper(II) by ultrahigh concentrations were shown to be present in chernozem soil. Microorganisms grew in the medium containing up to 500 mg/l Cu2+ (CuSO4 solution) and up to 10000 mg/l Cu2+ (in complex with citrate). Chelation of copper(II) with citrate was found to lead to increase of microbial resistance in 20 times. It was determined that a vanishingly small number of microorganisms resistant to toxic copper by ultrahigh concentrations can exist in a natural ecosystem. The number of viable microorganisms decreases with the increase in the content of Cu2+ that is described by the hyperbolic curve. Conclusions. The proposed methodological approach not only allows to isolate copper-resistant microorganisms from natural ecosystems of all geographic zones of the globe, but also avoid complex genetic transformations in order to obtain perspective genetically modified strains for further application in biotechnologies for purification of industrial wastewater. Keywords: copperresistant microorganisms, chernozem soil of Ukraine, diversified microbial community, environmental biotechnologies.