Features of low-temperature storage of streptomyces strains - producers of glycosidaz inhibitors

The aim of the work is to study the inhibitory activity of the Streptomyces lucensis VKPM Ac-1743 and Streptomyces violaceus VKPM Ac-1734 strains stored at –18 °C in a 15 % glycerol solution and in a 0.9 % sodium chloride solution.The object of the study was the actinomycete strains of S. lucensis VKPM Ac-1743 and S. violaceus VKPM Ac-1734 producers of glycosidase inhibitor, which is a micro-ingredient for creating products with a low glycemic index. The storage time was nine months. Bookmark storage was performed by flushing with a taped agar starchcontaining environment of Capek. For comparison, we studied the properties of cultures stored at +4 °C without cryoprotectant.Deep cultivation of Streptomyces strains was carried out in a periodic manner on a medium containing corn starch hydrolyzate under the Multitron incubator shaker (INFORS,Switzerland).Inhibitory activity was determined in inactivated native solutions by a colorimetric method with respect to pancreatic α-amylase, a test glycosidase involved in carbohydrate metabolism, and whose activity was chosen as a criterion for assessing the hypoglycemic action of the inhibitor. The proteinase activity of the inactivated native solution was determined by a modified method using a casein substrate.The experimental data were processed using the methods of mathematical statistics and Excel XP programs.As a result of the research, it was established that the actinomycete strains of S. lucensis and S. violaceus, the producers of glycosidase inhibitors, can maintain inhibitory activity during low-temperature storage for nine months.For the S. lucensis actinomycete strain, stored in a 15 % glycerol solution at – 18 °C, the maximum inhibitory activity is 48 hours in the biotechnological process and is (3686 ± 300) IU/cm3 native solution, and for the violaceus strain – (3150 ± 200) IU/cm³ of the native solution, respectively.For the strain of actinomycete S. lucensis, stored in 0.9 % sodium chloride solution at a temperature of – 18 °C, the maximum inhibitory activity accounts for 72 h of the biotechnological process and is (2600 ± 200) IU/cm³ of the native solution, and for the strain S. violaceus the maximum inhibitory activity accounts for 24 hours of the biotechnological process and is (3530 ± 200) IU/cm³ of the native solution.At a storage temperature of +4 °C, the inhibitory activity for the strain of actinomycete S. lucensis is (560 ± 20) IU/cm³ of native solution, and for the strain of S. violaceus – (1747 ± 100) IU/cm³ of native solution, respectively. On the basis of the data obtained, it can be concluded that a temperature of –18 °C is preferred for long-term storage.During the cultivation of Streptomyces strains, proteinase activity ranged from (0,012 ± 0,001) U/cm³ to (0,072 ± 0,002) U/cm³.The obtained data can be applied in further studies to develop conditions for long-term storage of collection crops.The aim of the work is to study the inhibitory activity of the Streptomyces lucensis VKPM Ac-1743 and Streptomyces violaceus VKPM Ac-1734 strains stored at –18 °C in a 15 % glycerol solution and in a 0.9 % sodium chloride solution.The object of the study was the actinomycete strains of S. lucensis VKPM Ac-1743 and S. violaceus VKPM Ac-1734 producers of glycosidase inhibitor, which is a micro-ingredient for creating products with a low glycemic index. The storage time was nine months. Bookmark storage was performed by flushing with a taped agar starchcontaining environment of Capek. For comparison, we studied the properties of cultures stored at +4 °C without cryoprotectant.Deep cultivation of Streptomyces strains was carried out in a periodic manner on a medium containing corn starch hydrolyzate under the Multitron incubator shaker (INFORS,Switzerland).Inhibitory activity was determined in inactivated native solutions by a colorimetric method with respect to pancreatic α-amylase, a test glycosidase involved in carbohydrate metabolism, and whose activity was chosen as a criterion for assessing the hypoglycemic action of the inhibitor. The proteinase activity of the inactivated native solution was determined by a modified method using a casein substrate.The experimental data were processed using the methods of mathematical statistics and Excel XP programs.As a result of the research, it was established that the actinomycete strains of S. lucensis and S. violaceus, the producers of glycosidase inhibitors, can maintain inhibitory activity during low-temperature storage for nine months.For the S. lucensis actinomycete strain, stored in a 15 % glycerol solution at – 18 °C, the maximum inhibitory activity is 48 hours in the biotechnological process and is (3686 ± 300) IU/cm3 native solution, and for the violaceus strain – (3150 ± 200) IU/cm³ of the native solution, respectively.For the strain of actinomycete S. lucensis, stored in 0.9 % sodium chloride solution at a temperature of – 18 °C, the maximum inhibitory activity accounts for 72 h of the biotechnological process and is (2600 ± 200) IU/cm³ of the native solution, and for the strain S. violaceus the maximum inhibitory activity accounts for 24 hours of the biotechnological process and is (3530 ± 200) IU/cm³ of the native solution.At a storage temperature of +4 °C, the inhibitory activity for the strain of actinomycete S. lucensis is (560 ± 20) IU/cm³ of native solution, and for the strain of S. violaceus – (1747 ± 100) IU/cm³ of native solution, respectively. On the basis of the data obtained, it can be concluded that a temperature of –18 °C is preferred for long-term storage.During the cultivation of Streptomyces strains, proteinase activity ranged from (0,012 ± 0,001) U/cm³ to (0,072 ± 0,002) U/cm³.The obtained data can be applied in further studies to develop conditions for long-term storage of collection crops

Beta-glucans from biomass of plant and microbial origin

The aim of the present study is to explore the transformation of (1→3)(1→4)-β-D-glucans of rye biomass by Aspergills niger and accumulation of (1→3)(1→6)-β-D-glucans in the microbial cell wall.Biomass from rye grain was obtained as a result of enzymatic hydrolysis of grain grinding of Omsk region of non-standard quality with grain impurity content of 45 ± 2 % by preparations (1→4)-β-glucanolytic, (1→3)-β-glucanolytic, (1→4)-xylanolytic and (1→4)-amylolytic action. Fermentation of hydrolysates, sucrosemineral and molasses medium by A. niger was carried out by a batch process under aerobic conditions. Determined the content of β-glucans, amino-nitrogen, glucose, disaccharides in grinding grain rye, rye biomass, the biomass of A. niger, the supernatants by colorimetric methods. Determination of chitin in biomass and qualitative determination of chitosan in supernatants of hydrolysates was carried out using chitosan sulfate sample and subsequent microscopy.The results of the research showed that (1→3)(1→4)-β-D-glucans in grain grinding are 10.2 ± 0.2 % in terms of dry matter, which exceeds the content of polysaccharide in the grain of standard quality by 1.5 – 3 times. In rye biomass revealed their smaller amount, 6.4 ± 0.5 %, apparently, due to the action of (1→4)and (1→3)-β-glucanase, (1→4)-xylanase and (1→4)-amylase. In microbial mass A. niger content of (1→3)(1→6)-β-Dglucans were at the level of 21.7 ± 0.7 %.On the basis of the obtained results, it was concluded that it is possible to use rye grain of non-standard quality, with a high content of grain impurities and a low proportion of starch polysaccharides, as a source of β-glucancontaining substrate for biosynthesis (1→3)(1→6)-β-D-glucans by A. niger having advantages over (1→3) (1→4)-β-D-glucans of plant origin. They are functionally more active and have a wide range of applications, namely as food additives in the manufacture of a wide range of products: for the enrichment of fibers, increasing the shelf life of products due to its water-binding properties, as thickeners, emulsifying and fat-reducing microingredients, stabilizers of creamy emulsions, textureformers, flavor enhancers.The aim of the present study is to explore the transformation of (1→3)(1→4)-β-D-glucans of rye biomass by Aspergills niger and accumulation of (1→3)(1→6)-β-D-glucans in the microbial cell wall.Biomass from rye grain was obtained as a result of enzymatic hydrolysis of grain grinding of Omsk region of non-standard quality with grain impurity content of 45 ± 2 % by preparations (1→4)-β-glucanolytic, (1→3)-β-glucanolytic, (1→4)-xylanolytic and (1→4)-amylolytic action. Fermentation of hydrolysates, sucrosemineral and molasses medium by A. niger was carried out by a batch process under aerobic conditions. Determined the content of β-glucans, amino-nitrogen, glucose, disaccharides in grinding grain rye, rye biomass, the biomass of A. niger, the supernatants by colorimetric methods. Determination of chitin in biomass and qualitative determination of chitosan in supernatants of hydrolysates was carried out using chitosan sulfate sample and subsequent microscopy.The results of the research showed that (1→3)(1→4)-β-D-glucans in grain grinding are 10.2 ± 0.2 % in terms of dry matter, which exceeds the content of polysaccharide in the grain of standard quality by 1.5 – 3 times. In rye biomass revealed their smaller amount, 6.4 ± 0.5 %, apparently, due to the action of (1→4)and (1→3)-β-glucanase, (1→4)-xylanase and (1→4)-amylase. In microbial mass A. niger content of (1→3)(1→6)-β-Dglucans were at the level of 21.7 ± 0.7 %.On the basis of the obtained results, it was concluded that it is possible to use rye grain of non-standard quality, with a high content of grain impurities and a low proportion of starch polysaccharides, as a source of β-glucancontaining substrate for biosynthesis (1→3)(1→6)-β-D-glucans by A. niger having advantages over (1→3) (1→4)-β-D-glucans of plant origin. They are functionally more active and have a wide range of applications, namely as food additives in the manufacture of a wide range of products: for the enrichment of fibers, increasing the shelf life of products due to its water-binding properties, as thickeners, emulsifying and fat-reducing microingredients, stabilizers of creamy emulsions, textureformers, flavor enhancers

Researching of features byosinthesis beta-glucans in the micelial mass Aspergillus niger and in non-conditional cereal grains

At the moment, it seems relevant to process mycelial mass as a waste product of citric acid and substandard grain, since there is a problem of recycling production waste. In turn, waste processing makes it possible to isolate a number of unique compounds, such as glucan and chitin. Of interest is the cell wall of micromycetes. Its main structural polysaccharides (chitin and glucan) form a complex called the chitin-glucan complex. Moreover, the content of chitin in micromycetes is higher than in traditional raw materials - shells of marine crustaceans. The mycelial mass of Aspergillus niger micromycete is a promising source of glucan and chitin. Aspergillus niger biomass and substandard cereal grains contain soluble dietary fiber-beta-glucans. They help reduce the risk of cardiovascular disease by lowering cholesterol, control blood glucose, which is very important for patients with diabetes. In addition, because of the ability of beta-glucans to form viscous gels, these chemicals slow down the absorption of sugars. Due to this, there is a decrease in the glycemic index of products. Of greatest interest are glucans of microbial origin. In addition to structural features (more functionally active ?-1,3 / 1,6-bonds), the advantage of beta-glucans derived from biomass is a higher quantitative yield of soluble forms, which are interesting not only in the food industry, but also in medicine . The aim of this work is to study the effect of various carbon sources (raw materials) on the biosynthesis of glucan-containing compounds and their derivatives Aspergillus niger micromycetes. As a result of studies, it was found that the biomass on the corn starch hydrolyzate is the most effective, it contains a high content of beta-glucans

Heterogeneous Distribution of Phospholipid Molecular Species in the Surface Culture of <i>Flammulina velutipes</i>: New Facts about Lipids Containing α-Linolenic Fatty Acid

Mycelial fungi grow as colonies consisting of polar growing hyphae, developing radially from spore or inoculum. Over time, the colony develops, hyphae are subject to various exogenous or endogenous stimuli, and mycelium becomes heterogeneous in growth, gene expression, biosynthesis, and secretion of proteins and metabolites. Although the biochemical and molecular mechanisms of mycelium heterogeneity have been the subject of many studies, the role of lipids in colony development and zonality is still not understood. This work was undertaken to extend our knowledge of mycelium heterogeneity and to answer the question of how different lipid molecular species are distributed in the surface colony of the basidial fungus Flammulina velutipes and how this distribution correlates with its morphology. The heterogeneity in the lipid metabolism and lipid composition of the fungal mycelium was demonstrated. According to the real-time PCR and LC-MS/MS results, the expression of genes of PC metabolism, accumulation of phospholipid classes, and degree of unsaturation of PC and PE increased in the direction from the center to the periphery of the colony. The peripheral zone of the colony was characterized by a higher value of the PC/PE ratio and a higher level of phospholipids esterified by linolenic acid. Considering that the synthesis of phospholipids in fungi occurs in different ways, we also conducted experiments with deuterium-labeled phospholipid precursors and found out that the Kennedy pathway is the predominant route for PC biosynthesis in F. velutipes. The zonal differences in gene expression and lipid composition can be explained by the participation of membrane lipids in polar growth maintenance and regulation

Diversity of ESI-MS Based Phosphatidylcholine Profiles in Basidiomycetes

Phosphatidylcholines (PC) are the main membrane lipid constituents comprising more than 50% of total glycerophospholipids. They coordinate a number of cell functions, particularly cell growth, homeostasis, secretion, recognition and communication. In basidial fungi PC are synthesized via the Kennedy pathway as well as through methylation of phosphatidylethanolamines (PE) and then undergo remodeling in Lands cycle that replaces fatty acids in PC molecules. The molecular profile of PC is determined by the genetic features that are characteristic for every species and depend on the environment. Here we present the results of ESI-MS based analyses of PC profiles of 38 species of basidiomycetes belonging to Agaricales (12), Polyporales (17), Russulales (5), Gleophyllales (2), Cantharellales (1), Auriculariales (1), Phallales (1). Although the variety of PC molecular species of basidiomycetes is rather diverse (20–38 molecular species in every profile), only 1–3 main molecular species represent 70–90% of total PC content. The most abundant of them are C36:4 and C36:3, followed by C34:1, C34:2, C36:5, C36:2. In the majority of basidiomycetes, C36:4 reaches up to 50–70% of total PC molecular species. Based on the results of hierarchical cluster analysis four main types of PC profiles which characterized the studied fungi independently from their taxonomic position, ecology, trophic status, and hyphal differentiation have been revealed. Comparative analyses of studied fungi using PCA method have shown that species of Polyporales differ from those of Agaricales by higher variability of PC profiles