Determination of fructan exohydrolase activity in the crude extracts of plants

Background: The roots of chicory and the tubers of Jerusalem artichoke are used for the production of inulin. However, a quality of tubers and roots, i.e. the content of inulin, monosaccarides and disaccharides, depends on the activity of enzymes implicated in the metabolism of inulin. The knowledge on the changes of activities of inulin synthesizing and degrading enzymes is limited during plant sprouting, growth and dormancy. It happens due to complicated measurements of the product of enzymatic reaction in the presence of crude plant extract. Fructan exohydrolase (\u3b2-D-fructan fructohydrolase, FEH, EC 3.2.1.80) is an enzyme responsible for the hydrolysis of fructans in plants. For fructose as the reaction product measurement, a high-performance liquid chromatography is usually used. The aim of the study was to choose a simple and suitable method for FEH activity determination and the measurement of fructose in the presence of plant extracts. Results: Two chemical methods, i.e. copper(II)\u2013neocuproine and 3,5-dinitrosalicylic acid, and the enzymatic one based on the reactions catalyzed by hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase were used. Enzymatic method was found to be suitable for FEH activity determination in plant extracts, and on the contrary to chemical methods no interference effects of compounds from crude plant extracts were observed. Conclusion: Enzymatic method is applicable for the routine analysis and will allow performing the investigations without special equipment on the inulin degrading enzyme in biotechnologically important crops

Correlation between Fructan Exohydrolase Activity and the Quality of Helianthus tuberosus L. Tubers

Jerusalem artichoke tubers have diverse applications in the food industry as well as in biotechnology. Their suitability depends mostly on the inulin content. Seasonal fluctuations of fructan exohydrolase activity responsible for inulin degradation was investigated in the tubers of three Jerusalem artichoke cultivars. The changes of fructan exohydrolase activity positively correlated with the changes of the content of total and short fructooligosaccharides. Therefore, to extract inulin with higher degree of polymerization for biotechnological purposes, the tubers of Jerusalem artichoke should be uprooted in autumn before the level of fructan exohydrolase reaches its maximum. If short fructooligosaccharides are desirable, the tubers in late autumn or spring tubers overwintered in soil are suitable

Inactivation of Escherichia coli Using Nanosecond Electric Fields and Nisin Nanoparticles: A Kinetics Study

Nisin is a recognized bacteriocin widely used in food processing, however, being ineffective against gram-negative bacteria and in complex food systems. As a result, the research of methods that have cell wall–permeabilizing activity is required. In this study, electroporation to trigger sensitization of gram-negative bacteria to nisin-loaded pectin nanoparticles was used. As a model microorganism, bioluminescent strain of E. coli was introduced. Inactivation kinetics using nanosecond pulsed electric fields (PEFs) and nisin nanoparticles have been studied in a broad range (100–900 ns, 10–30 kV/cm) of pulse parameters. As a reference, the microsecond range protocols (100 μs × 8) have been applied. It was determined that the 20–30 kV/cm electric field with pulse duration ranging from 500 to 900 ns was sufficient to cause significant permeabilization of E. coli to trigger a synergistic response with the nisin treatment. The kinetics of the inactivation was studied with a time resolution of 2.5 min, which provided experimental evidence that the efficacy of nisin-based treatment can be effectively controlled in time using PEF. The results and the proposed methodology for rapid detection of bacteria inactivation rate based on bioluminescence may be useful in the development and optimization of protocols for PEF-based treatments

ОБЛАСТЬ ПРИМЕНЕНИЯ ТЕМПЕРАТУРНЫХ ЕРД-МОДЕЛЕЙ ДИРОФИЛЯРИОЗА

Objective of research: To study the possibility of using temperature-based models for prevention of dirofilariasis. Materials and methods. For mathematical modeling of dirofilariasis we use the HDUstemperature model based on the impact of the average daily temperature on the rate of development of Dirofilaria larvae in mosquitoes.The amount of 130 DDU (Dirofilaria development units) accumulated in the period no more than 30 days at average daily temperature more than 14◦C is required for the development of Dirofilaria up to the infective stage.Daily data on average air temperature in Rostov-on-Don (1996 — 2012), Veliky Novgorod (2008 — 2012), Anapa (2008 -2012) and Astrakhan (2008 -2012) were used for the calculation.Results and discussion. The results of temperature simulation revealed that the DDU model is a low-priority forecasting model for canine dirofilariasis (Pearson’s correlation coefficient minus 0.45). The model considers only the average daily temperature and does not consider other factors affecting the incidence rates.The epidemic season of dirofilariasis in Rostov-on-Don in 1999 — 2012 differed depending on average daily temperatures. Therefore, the earliest date of the transmission of dirofilariasis in that period fell on the 12th of May, 2012; the latest date — on 29th of June, 2001. The optimal time for prevention of canine dirofilariasis has been defined (in Rostov-on-Don, Anapa and Astrakhan microfilaricides should be given to dogs from May 15 to November 15; in Veliky Novgorod — from June 15 to August 31).Thus, DDU-models (Dirofilaria Development Units) can be used to set time limits for epidemiological season of dirofilariasis and preventive treatment of dogs against dirofilaria.Цель исследования — изучить возможность применения температурной ЕРД-модели для профилактики дирофиляриоза.Материалы и методы. При математическом моделировании дирофиляриоза использована климатическая HDUs-модель, которая основана на влиянии среднесуточной температуры воздуха на скорость развития личинок дирофилярий в комарах. Для развития личинок до инвазионной стадии необходима сумма в 130 единиц развития дирофилярий (ЕРД), накопленная в срок не более 30 суток при среднесуточной температуре свыше 14 оC. Для расчета использованы ежедневные данные о среднесуточной температуре воздуха для городов Ростов-на-Дону (1996–2012 гг.), В. Новгород (2008–2012 гг.), Анапа и Астрахань (2008–2012 гг.).Результаты и обсуждение. По результатам температурного моделирования установлено, что ЕРД-модели имеют низкую значимость для прогнозирования заболеваемости собак дирофиляриозом (коэффициент корреляции Пирсона минус 0,45). Модель учитывает только среднесуточную температуру и не учитывает другие факторы, влияющие на показатели заболеваемости. Сроки эпидемического сезона дирофиляриоза в г. Ростове-на-Дону с 1999 по 2012 гг. отличались в зависимости от среднесуточных температур. Так, самая ранняя дата начала сезона передачи дирофиляриоза за этот период приходится на 2012 год — 12 мая, самая поздняя на 2001 год — 29 июня. Определены оптимальные сроки профилактики дирофиляриоза у собак: для гг. Ростова-на-Дону, Анапы, Астрахани дачу микрофилярицидов собакам необходимо продолжать с 15 мая по 4 ноября, в г. Великом Новгороде — с 15 июня по 31 августа. Таким образом, ЕРД-модели дирофиляриоза можно применять для установления сроков эпидемиологического сезона дирофиляриоза и сроков профилактической обработки собак от дирофиляриоза

Polietilenoksido šonines grandines turinčių polielektrolitų sintezė gyvybingosios radikalinės polimerizacijos metodais

The main goal of this work was to synthesize polyelectrolyte brushes containing poly(ethylene oxide) side chains by living polymerization. Brush-like macromolecules are unique polymer molecules whose conformation and physical properties are controlled by steric repulsion of densely grafted side chains. Molecules can be either flexible or stiff, depending on the grafting density and the length of the side chains. Polymerization of macromonomers is one of the most useful ways to prepare brush copolymers. However, with the macromonomer method, the distribution of the spacing of the side chains cannot be entirely controlled which along with broad and multimodal molecular weight distribution makes these polymers heterogeneous. These problems were considered to be overcome with the use of controlled/living polymerization. Conventional free-radical and RAFT copolymerization of poly(ethylene oxide) substituent containing methacrylate macromonomers, PEO5MEMA and PEO45MEMA, with methacrylic acid (MAA) was studied by the use of 1H NMR spectroscopy for an analysis of residual monomers. RAFT copolymerization of PEO45MEMA and MAA enabled to synthesize comb copolymers with low composition distribution and more homogeneous distribution of PEO side chains along the mainchain. Amphiphilic non-ionic blockcopolymers poly(lauryl methacrylate) (PLMA) – P(PEOnMEMA) and amphiphilic anionic blockcopolymers PLMA – PMAA were prepared by the RAFT method, and their properties in aqueous and THF solutions were studied. Cationic brush-on-brush polyelectrolytes with high density of PEO chains were prepared for the first time by ATRP method. Adsorption of novel cationic brush-on-brush polyelectrolytes on negatively charged silica surfaces was studied. The novel brush-on-brush polyelectrolytes are promising candidates for applications requiring good colloidal stability and they also are promising as lubricants in aqueous solutions

Determination of fructan exohydrolase activity in the crude extracts of plants

Background: The roots of chicory and the tubers of Jerusalem artichoke are used for the production of inulin. However, a quality of tubers and roots, i.e. the content of inulin, monosaccarides and disaccharides, depends on the activity of enzymes implicated in the metabolism of inulin. The knowledge on the changes of activities of inulin synthesizing and degrading enzymes is limited during plant sprouting, growth and dormancy. It happens due to complicated measurements of the product of enzymatic reaction in the presence of crude plant extract. Fructan exohydrolase (β-d-fructan fructohydrolase, FEH, EC 3.2.1.80) is an enzyme responsible for the hydrolysis of fructans in plants. For fructose as the reaction product measurement, a high-performance liquid chromatography is usually used. The aim of the study was to choose a simple and suitable method for FEH activity determination and the measurement of fructose in the presence of plant extracts. Results: Two chemical methods, i.e. copper(II)–neocuproine and 3,5-dinitrosalicylic acid, and the enzymatic one based on the reactions catalyzed by hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase were used. Enzymatic method was found to be suitable for FEH activity determination in plant extracts, and on the contrary to chemical methods no interference effects of compounds from crude plant extracts were observed. Conclusion: Enzymatic method is applicable for the routine analysis and will allow performing the investigations without special equipment on the inulin degrading enzyme in biotechnologically important crops

Synthesis of polyelectrolytes containing poly(ethylene oxide) side chains by living radical polymerization

The main goal of this work was to synthesize polyelectrolyte brushes containing poly(ethylene oxide) side chains by living polymerization. Brush-like macromolecules are unique polymer molecules whose conformation and physical properties are controlled by steric repulsion of densely grafted side chains. Molecules can be either flexible or stiff, depending on the grafting density and the length of the side chains. Polymerization of macromonomers is one of the most useful ways to prepare brush copolymers. However, with the macromonomer method, the distribution of the spacing of the side chains cannot be entirely controlled which along with broad and multimodal molecular weight distribution makes these polymers heterogeneous. These problems were considered to be overcome with the use of controlled/living polymerization. Conventional free-radical and RAFT copolymerization of poly(ethylene oxide) substituent containing methacrylate macromonomers, PEO5MEMA and PEO45MEMA, with methacrylic acid (MAA) was studied by the use of 1H NMR spectroscopy for an analysis of residual monomers. RAFT copolymerization of PEO45MEMA and MAA enabled to synthesize comb copolymers with low composition distribution and more homogeneous distribution of PEO side chains along the mainchain. Amphiphilic non-ionic blockcopolymers poly(lauryl methacrylate) (PLMA) – P(PEOnMEMA) and amphiphilic anionic blockcopolymers PLMA – PMAA were prepared by the RAFT method, and their properties in aqueous and THF solutions were studied. Cationic brush-on-brush polyelectrolytes with high density of PEO chains were prepared for the first time by ATRP method. Adsorption of novel cationic brush-on-brush polyelectrolytes on negatively charged silica surfaces was studied. The novel brush-on-brush polyelectrolytes are promising candidates for applications requiring good colloidal stability and they also are promising as lubricants in aqueous solutions

Correlation between Fructan Exohydrolase Activity and the Quality of Helianthus tuberosus L. Tubers

Jerusalem artichoke tubers have diverse applications in the food industry as well as in biotechnology. Their suitability depends mostly on the inulin content. Seasonal fluctuations of fructan exohydrolase activity responsible for inulin degradation was investigated in the tubers of three Jerusalem artichoke cultivars. The changes of fructan exohydrolase activity positively correlated with the changes of the content of total and short fructooligosaccharides. Therefore, to extract inulin with higher degree of polymerization for biotechnological purposes, the tubers of Jerusalem artichoke should be uprooted in autumn before the level of fructan exohydrolase reaches its maximum. If short fructooligosaccharides are desirable, the tubers in late autumn or spring tubers overwintered in soil are suitable

AREAS OF APPLICATION OF TEMPERATURE BASED DDU MODELS FOR PREVENTION OF DIROFILARIASIS

Objective of research: To study the possibility of using temperature-based models for prevention of dirofilariasis. Materials and methods. For mathematical modeling of dirofilariasis we use the HDUstemperature model based on the impact of the average daily temperature on the rate of development of Dirofilaria larvae in mosquitoes.The amount of 130 DDU (Dirofilaria development units) accumulated in the period no more than 30 days at average daily temperature more than 14◦C is required for the development of Dirofilaria up to the infective stage.Daily data on average air temperature in Rostov-on-Don (1996 — 2012), Veliky Novgorod (2008 — 2012), Anapa (2008 -2012) and Astrakhan (2008 -2012) were used for the calculation.Results and discussion. The results of temperature simulation revealed that the DDU model is a low-priority forecasting model for canine dirofilariasis (Pearson’s correlation coefficient minus 0.45). The model considers only the average daily temperature and does not consider other factors affecting the incidence rates.The epidemic season of dirofilariasis in Rostov-on-Don in 1999 — 2012 differed depending on average daily temperatures. Therefore, the earliest date of the transmission of dirofilariasis in that period fell on the 12th of May, 2012; the latest date — on 29th of June, 2001. The optimal time for prevention of canine dirofilariasis has been defined (in Rostov-on-Don, Anapa and Astrakhan microfilaricides should be given to dogs from May 15 to November 15; in Veliky Novgorod — from June 15 to August 31).Thus, DDU-models (Dirofilaria Development Units) can be used to set time limits for epidemiological season of dirofilariasis and preventive treatment of dogs against dirofilaria