Том 60, № 1 (2024)

The work demonstrates that recombinant CRISPR-nuclease Cas12a, purified after heterologous expression with a simplified method using single-stage metal-chelate chromatography, can be successfully utilized in DETECTR technology. The combination of CRISPR-nuclease Cas12a obtained by such way with recombinase polymerase amplification (RPA) allowed one to ensure the selectivity of detection of Dickeya solani — the dangerous bacterial phytopathogen causing the potato disease known as “blackleg” — against closely related and unrelated bacterial phytopathogens with a limit of detection of 1 copy of the bacterial genome per amplification reaction. The result can be determined visually, without the use of complex instrumental methods, by changing the color of the reaction sample when illuminated with blue light that creates the basis for development of field DNA diagnostics of D. solani. The use of simplified chromatographic purification will significantly reduce the time and resources required to obtain a functionally active CRISPR-nuclease Cas12a for development and production of DNA diagnostics based on DETECTR technology.

In this work, oxidative damage and the level of antioxidant response in Acinetobacter calcoaceticus, Pseudomonas putida, and Rhodococcus erythropolis cells under the influence of such antibiotics as ampicillin, azithromycin, rifampicin, tetracycline, and ceftriaxone were studied. The level of protein carboxylation and lipid peroxidation (LPO), as well as the activity of superoxide dismutase (SOD), catalase, glutathione reductase (GR), and the level of glutathione 3 and 6 hours after antibiotic treatment of bacteria were assessed. It is observed that SOD induction occurs earlier and is more active than catalase induction. In A. calcoaceticus, SOD is induced together with protein carboxylation and probably protects them from oxidative damage, while catalase induction correlates with LPO. A positive correlation is also noted between catalase activity and glutathione content in R. erythropolis. Catalase activity increases insignificantly and even decreases under the studied antibiotics influence, which is associated with an insignificant level of lipid peroxidation in most prokaryotes. On the other hand, low catalase activity can contribute to genome destabilization as a result of oxidative stress and enhance the adaptive evolution of bacteria.

The dose-dependent effect of the A. brasilense Sp7 lectin on the roots of 4-day-old wheat seedlings (Triticum aestivum L. cv. Saratovskaya 29) grown under simulated salt stress was studied. In the roots of wheat seedlings under salt stress, lectin increased the activity of peroxidase and superoxide dismutase, but decreased the activity of catalase. In the roots of stressed seedlings, lectin reduced the total protein content and lipid peroxidation causing membrane damage, but increased the content of secondary metabolites, such as the total amount of phenols and flavonoids. It was concluded that azospirillum lectins are involved in adaptive changes in the roots of wheat seedlings, due to which the relationship between bacteria and their hosts can be regulated when soil and climatic factors change.

The effect of four enzyme preparations: bacillolysin, agroprot, protozyme and protozyme C (Russia) on solubility, emulsifying activity, emulsion stability, foaming and foam stability of isolates preparated from two varieties of peas was studied. It is shown that treatment with enzymes can increase the solubility of isolates at pH 5 by more than 7 times, the index of emulsifying activity at pH 5 by 1.5 to 2 times, and at pH 6 by almost 1.5 times; the stability index of the emulsion increased by about 20% at pH 5, and by 1.7 times (in one of the varieties) at pH 6; foaming increased by 2.4 to 3 times at pH 5, and at pH 6 by 1.8 to 3.7 times; foam stability increased by 25 to 33% at pH 5 and by more than 1.5 times (in one of the varieties) at pH 6. The results obtained made it possible to select an enzyme preparation (bacterial alkaline serine protease) to improve the parameters of pea protein isolates intended for the manufacture of analogues of fermented milk products.

An analysis of the biological value of chum salmon showed that, in terms of the balance of the amino acid composition of the protein, chum salmon with spawning changes is not inferior to chum salmon without spawning changes (chum salmon “silver”). A higher content of polyunsaturated fatty acids in total lipids, including omega-3, was noted in chum salmon with spawning changes compared to chum salmon “silver”. The metabolic profile of chum salmon samples obtained by NMR spectroscopy showed that the levels of Asp, Glu and Glc markedly increase during spawning migration, which is explained by the limited feeding of fish. The total taste index was calculated of chum salmon «silver», which was 4.06 ± 0.11, and that chum salmon with spawning changes was 3.46 ± 0.09, therefore, according to taste sensations chum salmon «silver» has a richer taste compared to chum salmon with spawning changes. The results of the analysis of the nutritional value and metabolic profile of chum salmon with spawning changes can be used in the manufacture of minced fish for the subsequent production of specialized food products by introducing functional additives that balance the consistency, color, and also give a functional orientation.

The influence of thermodynamic and kinetic conditions on the interaction of polyclonal antibodies to penicillins with the antibiotics of a penicillin group was studied in the system of a direct enzyme-linked immunosorbent assay (ELISA). Minimum differences in the cross reactions of the polyclonal antibodies with different penicillins were observed when the ELISA was carried out at 4°C for 1 hour. An increase in temperature and duration of the assay led to an increase in antibodies reactivity only to amoxicillin, and significantly enhanced differences among the sensitivities of individual penicillins determination. Under the chosen assay conditions, the following antibodies cross-reactivity values were obtained: to penicillin G — 90%, to ampicillin — 100%, to amoxicillin — 110%. The analytical sensitivity was 0.03 ng/mL for ampicillin, and the limit of ampicillin quantification in milk was 0.4 μg/L. The developed group-specific ELISA was used for the determination in milk of seven penicillins that are regulatory controlled in foods and raw materials of animal origin — penicillin G, ampicillin, amoxicillin, cloxacillin, oxacillin, dicloxacillin and nafcillin.