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Separating amphiphilic compounds from complex matrix is challenging. Surfactin, cyclic lipopeptides group, was used as a model to find the proper separation conditions and parameters. The aim of the work was to solve the emulsification plug problem in countercurrent partition chromatography (CPC). The selected solvent system was a composition of n-heptane/n-butanol/methanol/aqueous buffer (20 mM disodium phosphate with 50 mM NaCl) at a ratio of 2:3:2:3. Several elution method modifications were applied. The most important was an appropriate mobile phase flow rate adjustment (flow rate gradient), minimizing the risk of stationary phase leakage. During the CPC procedures, the hydrostatic pressure was monitored as a factor of stationary phase retention. The hydrostatic pressure indicated the biphasic system’s stability. A flow rate of 2 ml/min resulted in column stabilization and peak resolution during chromatographic separation. In order to avoid uncontrolled leakage, the flow rate was increased gradually. At 3.5 ml/min, there was a small loss of the stationary phase as the hydrostatic pressure decreased from 32 to nearly 18 bar. After designing the method, it was tested with SU containing extracts obtained from SSF to show its effectiveness. Our results showed that the single dual-mode method can be used to remove matrix’s impurities and separate surfactin as a mixture of homologues with more than 80% purity. Moreover, application of the double dual-mode method allows for the isolation of fractions containing individual surfactin homologues in amounts of about 70% of the sample, which makes it possible to conduct further biological research.

BackgroundBacteriophages from the Bastillevirinae subfamily have proven effective against bacteria from the Bacillus genus including organisms from B. cereus group which causes food poisoning and persistent contamination of industrial installations. However, successful application of these phages in biocontrol depends on understanding of their biology and stability in different environments.MethodsIn this study we isolated a novel virus from the garden soil in Wrocław (Poland) and named it Thurquoise. Genome of the phage was sequenced using Illumina technology and assembled as a single continuous contig that represents consensus result of different assembly algorithms. Its morphology was determined using Cryo-EM imaging while the dynamics of replication by turbidimetric lysis assay. We also determined phage host range in the efficiency of plating (EOP) assay. Finally, the stability of the Thurquoise was tested by long term incubation in buffers containing different ions (Mg2+, Ca2+, Mn2+, Zn2+, Cu2+, K+, Co2+, Fe3+, Cs+) and freezing with various cryoprotectants (glycerol, gelatin, saccharose, trehalose).ResultsWe present a complete, carefully annotated genome of the Thurquoise phage with 226 identified protein genes and 18 tRNAs. The complex virion structure of this phage is typical for the Bastillevirinae family. Confirmed hosts include selected bacteria from the Bacillus cereus group - specifically B. thuringiensis and B. mycoides. Latent and eclipse periods of Thurquoise in the isolation host last ~40 min and ~50 min respectively. The phage remains viable for more than 8 weeks in variants of SM buffer with magnesium, calcium, cesium, manganese or potassium and can withstand numerous freeze-thaw cycles if protected by the addition of 15% glycerol or, to a lesser extent, 2% gelatin.ConclusionThe Thurquoise phage is the exemplar of the new candidate species in Caruleovirus genus in the Bastillevirinae subfamily of the Herelleviridae family with genome, morphology and biology typical for these taxa.With proper buffer formulation, this virus (and likely related phages) can be safely stored in common freezers and refrigerators for a considerable time.

Opportunistic pathogen

The ban on the use of zinc oxide has increased interest in probiotics, prebiotics, synbiotics and organic acids, as well as fermented components in the diet of weaned piglets. This study assessed the effect of 8% fermented rapeseed meal in weaner diets on characteristics of the gastrointestinal tract, the small intestinal microbiota, and immune and antioxidant status. The effects were determined by measuring biochemical and haematological blood parameters, levels of class G, A and M immunoglobulins and IL-6, and the antioxidant potential of the plasma. After slaughter, the gastrointestinal tract was measured, the viscosity of the digesta was determined, and microbiological tests were performed. The results showed that the fermented component reduced the viscosity of the digesta and the length of segments of the gastrointestinal tract. It caused a statistically significant increase in lactic acid bacteria and a decrease in total bacteria. The haematological and biochemical analyses of the blood confirmed the biological activity of the fermented component. Pigs from group FR had significantly higher haemoglobin levels (p = 0.001), RBC count (p = 0.015), and haematocrit (Ht) value (p Bacillus subtilis strain 87Y benefits gastrointestinal function by stabilizing and improving the function of the bacterial microbiota, inhibiting growth of certain pathogens, and strengthening immunity.

Bacillus subtilis strain 87Y, isolated from the earthworm Eisenia fetida, decreases the growth of pathogenic Salmonella spp. and Staphylococcus aureus and promotes the growth of probiotic Lactococcus spp. Preserving viability in acidic conditions as well as in bile salts, B. subtilis 87Y meets two of the requirements of a probiotic strain. Thanks to the production of the biosurfactant surfactin, B. subtilis 87Y limits the growth of the Gram-positive bacterium S. aureus. In the presence of sucrose, B. subtilis produces levan, which contributes to promoting the growth of other probiotics. Our in vitro studies justify the continuation of enriching rapeseed meal waste from solid-state fermentation with B. subtilis 87Y, to produce high-value animal feed supplements.

The traditional method of biodiesel production is based on the transesterification of triglycerides using an alkaline catalyst dissolved in methanol. The aim of this study was to replace a homogeneous alkaline catalyst with a heterogeneous catalyst on the carbon support. The use of a carbon enables the catalyst to be reusable in the production process, eliminates the formation of soaps and increases the glycerol purity. Fatty acid methyl esters were obtained from the transesterification of corn oil using KOH supported on activated carbon (KOH/AC). The effect of the molar ratio of methanol to oil, reaction time and catalyst amount were used to optimize the transesterification reaction. The optimum condition for waste corn oil transesterification to methyl ester was obtained below 0.75 wt.% catalyst amount. The yield was up to 92 wt.% at 62.5 °C, 1 h reaction time and 3:1 methanol-to-oil ratio. This study demonstrated that the transesterification of the waste corn oil using methanol can be effectively catalyzed by the developed catalyst.

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.

The specific structure and composition of the cell plasma membrane (PM) is crucial for many cellular processes and can be targeted by various substances with potential medical applications. In this context, biosurfactants (BS) constitute a promising group of natural compounds that possess several biological functions, including anticancer activity. Despite the efficiency of BS, their mode of action had never been elucidated before. Here, we demonstrate the influence of cyclic lipopeptide surfactin (SU) on the PM of CHO-K1 cells. Both FLIM and svFCS experiments show that even a low concentration of SU causes significant changes in the membrane fluidity and dynamic molecular organization. Further, we demonstrate that SU causes a relevant dose-dependent reduction of cellular cholesterol by extracting it from the PM. Finally, we show that CHO-25RA cells characterized by increased cholesterol levels are more sensitive to SU treatment than CHO-K1 cells. We propose that sterols organizing the PM raft nanodomains, constitute a potential target for SU and other biosurfactants. In our opinion, the anticancer activity of biosurfactants is directly related with the higher cholesterol content found in many cancer cells.

This study examined the influence of fermented rapeseed meal (FRSM) on the intestinal morphology and gut microflora of broiler chickens. Limited information is available on the effects of FRSM on the intestinal morphology and the gastrointestinal microbiome population of animals. First, 48 21-day Ross 308 broilers were placed in metabolic cages and randomly assigned to four experimental groups. Group I birds were negative controls and received no additive. Group II birds were positive controls and received a 3% addition of unfermented rapeseed meal. Group III birds received a 3% addition of rapeseed meal fermented with the Bacillus subtilis 67 bacterial strain. Group IV birds received a 3% addition of rapeseed meal fermented with the B. subtilis 87Y strain. After 23 days of experimental feeding, the contents of the birds’ ceca were collected for microorganism determination. The histomorphology of the broilers’ ceca was also determined, and beneficial changes were found in the histology of the broilers’ ceca with the additives. Moreover, these materials inhibited the growth of pathogens and significantly stimulated the growth of probiotic bacteria. These results suggest that the addition of 3% FRSM has a potential probiotic effect and can be used as a material in feed for broilers.

The developing field of bio-nanotechnology aims to advance colloidal research via the introduction of multifunctional nanoparticles to augment the dermal effectiveness of active substances. Self-emulsifying drug delivery systems (SEDDS)—isotropic mixtures of oils, surfactants, solvents and co-solvents or surfactants—are attracting interest in the cosmeceutical field. As part of this study, SEDDS systems containing vitamin C or vitamin E and curcumin were developed, whereby the bioavailability of the active compounds increased by enhancing their permeability to deeper layers of the skin. A composition consisting of 50% surfactin from Bacillus subtilis, 30% Transcutol and 20% oil phase was designed to encapsulate the active substances, i.e., vitamin C or vitamin E and curcumin, contained in the oil phase. The developed carriers were characterized by average particle sizes of 69–183 nm. The formulations with the vitamins were found to be physically and chemically stable for 6 months. Transdermal tests were carried out, showing that the carriers enable the transport of active substances deep into the skin, stopping at the dermis border. The formulations with vitamin C and vitamin E reduced the discoloration, the vascular lesions, and the depth of the wrinkles on the tested skin, which can be useful in cosmetics in the treatment of problem skin, including capillary and sensitive skin.

The technological problems occurring in the co-firing of biomass and brown coal (lignite) prompted this research project. During the fuel preparation, accidental self-ignition and explosions were several times reported by power plants operators. The aim of this study was to evaluate brown coal, sunflower husks and sunflower husk pellets as fuels for co-firing in energetic boilers. Sunflower husk had a lower ash content and calorific value than the pellets. The range of the combustion temperatures of the biomass (200–300 °C) was narrower than that of brown coal (200–800 °C). The formation of highly alkaline ash from the biomass resulted in the formation in boiler of agglomerates of ash. The elemental composition, thermogravimetric and biological analyses suggested that the pellets contained synthetic additives difficult to identify. The biological method was proposed for evaluating biomass additives. The use of additional agents in the pelletizing process may influence on the combustion parameters. Mixing biomass with brown coal may occasionally result in self-ignition in the logistic chain. Plastic additives and biological activity may contribute to self-ignition.

Pseudomonas aeruginosa is a common human pathogen belonging to the ESKAPE group. The multidrug resistance of bacteria is a considerable problem in treating patients and may lead to increased morbidity and mortality rate. The natural resistance in these organisms is caused by the production of specific enzymes and biofilm formation, while acquired resistance is multifactorial. Precise recognition of potential antibiotic resistance on different molecular levels is essential. Metabolomics tools may aid in the observation of the flux of low molecular weight compounds in biochemical pathways yielding additional information about drug-resistant bacteria. In this study, the metabolisms of two P. aeruginosa strains were compared—antibiotic susceptible vs. resistant. Analysis was performed on both intra- and extracellular metabolites. The 1H NMR method was used together with multivariate and univariate data analysis, additionally analysis of the metabolic pathways with the FELLA package was performed. The results revealed the differences in P. aeruginosa metabolism of drug-resistant and drug-susceptible strains and provided direct molecular information about P. aeruginosa response for different types of antibiotics. The most significant differences were found in the turnover of amino acids. This study can be a valuable source of information to complement research on drug resistance in P. aeruginosa.

The aim of the study was to determine effects of three levels of rapeseed meal (4% - FR4, 8% - FR8, and 12% - FR12) which has undergone fermentation and drying processes (FRSM) in the diet of crossbred rabbits (New Zealand White × Popielno White, n=40) on their gastrointestinal tract morphometry, internal organs weight, and proximate composition and physicochemical properties of skeletal muscles (biceps femoris – BF, and joined longissimus thoracis and longissimus lumborum – LTL). Although the total length of the digestive tract of rabbits did not differ significantly, the addition of FRSM was found to significantly (P The content of proximate chemical constituents in rabbit muscle tissues was similar, with except of collagen percentage. A larger share of FRSM (from 4% to 12%) was accompanied by an increased level of collagen in LTL (P

Drought and pest resistance, together with high oil content in its seeds, make Jatropha curcas a good oil source for biodiesel. Oil cake from J. curcas is not suitable for animal feeding and thus may be profitably used for additional energy production by conversion into biogas; however, the anaerobic digestion process must be optimized to obtain good efficiency. We subjected oil cake to thermal and acidic pretreatment to deactivate protease inhibitors and partially hydrolyze phytate. We then digested the samples in batch conditions to determine the effects of pretreatment on biogas production. Thermal pretreatment changed the kinetics of anaerobic digestion and reduced protease inhibitor activity and the concentration of phytate; however, biogas production efficiency was not affected (0.281 m 3 kg −1 ). To evaluate the possibility of recirculating water for SSF hydrolysis, ammonium nitrogen recovery from effluent was evaluated by its precipitation in the form of struvite (magnesium ammonium phosphate).Concentration of ammonium ions was reduced by 53% (to 980 mg L −1 ). We propose a water-saving concept based on percolation of J. curcas cake using anaerobic digestion effluent and feeding that percolate into a methanogenic bioreactor.

The rapid and accurate quantification of biosurfactants in biological samples is challenging. In contrast to the orcinol method for rhamnolipids, no simple biochemical method is available for the rapid quantification of lipopeptides. Various liquid chromatography (LC) methods are promising tools for relatively fast and exact quantification of lipopeptides. Here, we report strategies for the quantification of the lipopeptides pseudofactin and surfactin in bacterial cultures using different high- (HPLC) and ultra-performance liquid chromatography (UPLC) systems. We tested three strategies for sample pretreatment prior to LC analysis. In direct analysis (DA), bacterial cultures were injected directly and analyzed via LC. As a modification, we diluted the samples with methanol and detected an increase in lipopeptide recovery in the presence of methanol. Therefore, we suggest this simple modification as a tool for increasing the accuracy of LC methods. We also tested freeze-drying followed by solvent extraction (FDSE) as an alternative for the analysis of "heavy" samples. In FDSE, the bacterial cultures were freeze-dried, and the resulting powder was extracted with different solvents. Then, the organic extracts were analyzed via LC. Here, we determined the influence of the extracting solvent on lipopeptide recovery. HPLC methods allowed us to quantify pseudofactin and surfactin with run times of 15 and 20 min per sample, respectively, whereas UPLC quantification was as fast as 4 and 5.5 min per sample, respectively. Our methods provide highly accurate measurements and high recovery levels for lipopeptides. At the same time, UPLC-MS provides the possibility to identify lipopeptides and their structural isoforms.

The study aimed to assess whether Pseudomonas aeruginosa strains from different sources can be distinguished by the metabolomic fingerprint and to check whether antibiotic susceptibility distinctions are available through metabolomic analysis.

Abstract Pseudomonas aeruginosa is a common, Gram-negative environmental organism. It can be a significant pathogenic factor of severe infections in humans, especially in cystic fibrosis patients. Due to its natural resistance to antibiotics and the ability to form biofilms, infection with this pathogen can cause severe therapeutic problems. In recent years, metabolomic studies of P. aeruginosa have been performed. Therefore, in this review, we discussed recent achievements in the use of metabolomics methods in bacterial identification, differentiation, the interconnection between genome and metabolome, the influence of external factors on the bacterial metabolome and identification of new metabolites produced by P. aeruginosa. All of these studies may provide valuable information about metabolic pathways leading to an understanding of the adaptations of bacterial strains to a host environment, which can lead to new drug development and/or elaboration of new treatment and diagnostics strategies for Pseudomonas. Graphic abstract

Simple Summary The high protein requirement in the diet of mink is currently met using extruded cereals, meat and bone meal, which raises the cost of mink farming. At the same time, there are waste products that may contain many infectious agents. Therefore, there is a need to look for high-protein feeds with properties stabilizing the gastrointestinal tract (GIT). Fermented rapeseed meal (FRSM) may prove to be such a material. During the fermentation process, rapeseed loses the anti-nutritional substances contained in it, while acquiring new properties of a bioproduct. A study was carried out in mink to determine how FRSM in the diet modifies the population of microorganisms inhabiting the GIT and stabilizes its microbiota. FRSM was included in the feed of experimental animals, and changes in the population size of groups of intestinal microorganisms were analysed in relation to the control group receiving standard feed. Microbiological analyses were performed on faeces samples and from intestinal contents collected after the animals had been euthanized. The paper presents the effect of supplementation with FRSM obtained using biotechnological methods and demonstrates that it can be used in animal diets as a bioproduct influencing the gastrointestinal microbiota. Abstract Fermented rapeseed meal (FRSM) was used in the diet of American mink (Neovison vison). An advantage of this product is its prebiotic and functional properties, which can modify the bacterial microbiota of the GIT. A control group and three experimental groups were formed, with 60 animals in each group. The control group received a basal diet and the experimental groups received a diet with a 2%, 4% or 6% of FRSM as a replacement of extruded wheat. Bacillus subtilis strain 87Y was used to ferment the rapeseed meal (RSM). The study was conducted on mink from the age of 16–17 weeks until slaughter. Changes in the microbiota were analysed in samples of the animals’ faeces and intestinal contents. The analyses included determination of the total number of bacteria and fungi, the number of coliforms and Escherichia coli, the total number of anaerobic Clostridium perfringens, and the presence of Salmonella spp. In animals receiving 4% and 6% FRSM (groups II and III), the content of microscopic fungi and the number of C. perfringens bacteria was significantly (p ≤ 0.05) lower than in the animals from the control group (group 0). A decrease in E. coli was observed in all experimental groups (I, II and III), although these differences were not statistically significant. The inclusion of FRSM in the feed ration did not affect the number of lactic acid intestinal bacteria. Analysis of the results obtained from the stool samples showed that the inclusion of FRSM in the ration did not significantly affect the number of microorganisms in each group. However, as in the case of the intestinal contents, in these samples there was a decrease in the total number of C. perfringens in the experimental groups (I, II and III), with a simultaneous increase in the number of mesophilic bacteria in relation to the control. There was no detection of Salmonella bacteria in any of the analysed material.

Microbiologically produced surface acting compounds (biosurfactants) have very interesting properties with many potential industrial applications. Lipopeptides is a particularly promising group of biosurfactants in respect to the potentially huge number of various chemical structures. The chemical diversity results from fatty acid moiety (e.g. length, saturation, branching or hydroxylation) and type and sequence of the amino acids in the peptide chain. The limiting factor for the design and analysis of various lipopeptides is the ability of the targeted biosynthesis. Biosynthesis of particular lipopeptides may be potentially achieved by strain selection, culture conditions, or molecular engineering. The well-known lipopeptedes (surfactins, iturins, and fengycins) producer is B. subtilis . The aim of this study was to study targeted surfactin structural analogues biosynthesis in response to culture conditions in view of the design and production of tailor-made lipopeptides. Two B. subtilis strains (KB1 and #309) were tested for surfactin production. Both strains produced a mixture of five major surfactin analogues with the number of carbons in an alkyl chain ranging from 12 to 16. The two strains differed with respect to their oxygen demand for optimal surfactin biosynthesis (lower oxygen demand for KB1). The amount of air influenced the relative ratios of surfactin analogues. Lower oxygen amount decreased the share of C15 analogues while it increased the share of C12 analogues. Thus, the biosynthesis of a desired surfactin analogue may controlled by both strain and culture conditions.

The study aimed to assess whether Pseudomonas aeruginosa strains from different sources can be distinguished by the metabolomic fingerprint and to check whether antibiotic susceptibility distinctions are available through metabolomic analysis. 1H NMR spectroscopy analysis of the bacteria metabolites was performed. Twenty-nine strains were tested (18 isolated form cystic fibrosis patients and 11 environmental). Thirty-one metabolites were identified, 12 were up-regulated in strains from CF patients, while 2 were higher level in strains from the environment. Changed carbohydrate catabolic metabolism and the metabolic shift toward the utilization of amino acids is suggested in strains from CF patients.

Recognizing the &beta, glucan component of the Candida albicans cell wall is a necessary step involved in host immune system recognition. Compounds that result in exposed &beta, glucan recognizable to the immune system could be valuable antifungal drugs. Antifungal development is especially important because fungi are becoming increasingly drug resistant. This study demonstrates that lipopeptide, surfactin, unmasks &beta, glucan when the C. albicans cells lack ergosterol. This observation also holds when ergosterol is depleted by fluconazole. Surfactin does not enhance the effects of local chitin accumulation in the presence of fluconazole. Expression of the CHS3 gene, encoding a gene product resulting in 80% of cellular chitin, is downregulated. C. albicans exposure to fluconazole changes the composition and structure of the fungal plasma membrane. At the same time, the fungal cell wall is altered and remodeled in a way that makes the fungi susceptible to surfactin. In silico studies show that surfactin can form a complex with &beta, glucan. Surfactin forms a less stable complex with chitin, which in combination with lowering chitin synthesis, could be a second anti-fungal mechanism of action of this lipopeptide.

Levan is a polysaccharide composed of fructose units with β-2,6-glycoside bonds. Microorganisms synthesize levan by levansucrase as a mixture of low- and high-molecular-weight fractions. Due to its properties, it has a wide range of applications in cosmetics, pharmaceuticals, food and medicine; it appears that the molecular weight of levan might impact its industrial use. To obtain one fraction of levan after biotransformation, ethanol precipitation with an increasing volume of alcohol was conducted. This precipitation process was also optimized. Several types of analyses were used. Low-molecular-weight levan was evaluated for toxicity in a normal human dermal fibroblast cell line and hemolytic potential on human erythrocytes. Levan was found to be non-cytotoxic and non-hemolytic in concentrations ranging from 0.01 to 1.00 mg/ml. Moreover, levan demonstrated antioxidant potential expressed as an ability to inhibit of oil/water emulsion oxidation and DPPH radical scavenging.

Surfactin is obtained through biocatalysis by microorganisms. In our biorefinery concept, it is purified on activated carbon (AC) during downstream processing. Besides cyclic surfactin, it is possible to obtain linear surfactin analogues, when AC with specific properties is used. In the present article, the hydrolysis of various cyclic surfactin analogues mediated by activated carbon is described. Hydrolysis products were identified using HPLC/UV/MS and (Q-TOF)MS/MS. Hydrolytic activity of six commercial and three modified activated carbons was evaluated. The porous texture of ACs was determined by sorption measurements and elemental composition of ACs surface - by SEM-EDS analysis. Their pHPZC value and moisture, ash, and volatile matter content using proximate analysis were also determined. Properties of ACs were correlated with their hydrolytic activity, and the crucial role of alkaline pHPZC was found. The beneficial effect of alkaline pHPZC was further confirmed by acid modification of AC that had previously shown hydrolytic activity and lost this ability after the pHPZC decrease.

After extracting the oil from rapeseed, the remaining meal byproduct is used in animal feed, particularly for cattle, and represents an effective, high-protein substitute for soybean meal. The biotransformation of rapeseed meal using Generally Recognized as Safe (GRAS) bacteria increases its nutritional value and enriches it with a variety of additives including polymers, biosurfactants, and enzymes. Polymers produced in SSF process with rapeseed meal (e.g., levan) have probiotic prosperities and can even serve as alternatives to antibiotics, which are banned from animal feed by law. Due to their moisturizing properties, these polymers are also incorporated into cosmetics. The biosurfactants produced by bacteria and yeast confer their strong antimicrobial effects to preserve the feed. In turn, the many enzymes produced during the biotransformation of rapeseed meal increase its nutritional value by reducing fibers, detrimental substances (e.g., tannins, erucic acid, phytic acid), and mycotoxins. Taken together, rapeseed meal biotransformation results in numerous benefits, for the animal and industry alike.

Global warming and the reduction in our fossil fuel reservoir have forced humanity to look for new means of energy production. Agricultural waste remains a large source for biofuel and bioenergy production. Flax shives are a waste product obtained during the processing of flax fibers. We investigated the possibility of using low-lignin flax shives for biogas production, specifically by assessing the impact of CAD deficiency on the biochemical and structural properties of shives. The study used genetically modified flax plants with a silenced CAD gene, which encodes the key enzyme for lignin synthesis. Reducing the lignin content modified cellulose crystallinity, improved flax shive fermentation and optimized biogas production. Chemical pretreatment of the shive biomass further increased biogas production efficiency.

The methanogenic archaea are a group of micro-organisms that have developed a unique metabolic pathway for obtaining energy. There are 150 characterized species in this group; however, novel species continue to be discovered. Since methanogens are considered a crucial part of the carbon cycle in the anaerobic ecosystem, characterization of these micro-organisms is important for understanding anaerobic ecology. A methanogens database (MDB; http://metanogen.biotech.uni.wroc.pl/), including physiological and biochemical characteristics of methanogens, was constructed based on the descriptions of isolated type strains. Analysis of the data revealed that methanogens are able to grow from 0 to 122 °C. Methanogens growing at the same temperature may have very different growth rates. There is no clear correlation between the optimal growth temperature and the DNA G+C content. The following substrate preferences are observed in the database: 74.5 % of archaea species utilize H2+CO2, 33 % utilize methyl compounds and 8.5 % utilize acetate. Utilization of methyl compounds (mainly micro-organisms belonging to the genera Methanosarcina and Methanolobus ) is seldom accompanied by an ability to utilize H2+CO2. Very often, data for described species are incomplete, especially substrate preferences. Additional research leading to completion of missing information and development of standards, especially for substrate utilization, would be very helpful.

Development of balanced community of microorganisms is one of the obligatory for stable anaerobic digestion. Application of mathematical models might be helpful in development of reliable procedures during the process start-up period. Yet, the accuracy of forecast depends on the quality of input and parameters. In this study, the specific anaerobic activity (SAA) tests were applied in order to estimate microbial community structure. Obtained data was applied as input conditions for mathematical model of anaerobic digestion. The initial values of variables describing the amount of acetate and propionate utilizing microorganisms could be calculated on the basis of SAA results. The modelling based on those optimized variables could successfully reproduce the behavior of a real system during the continuous fermentation.

Polymer composites with natural fibers prepared by melt blending were investigated. Synthetic and natural macromolecules were used, including poly(lactic acid), polyhydroxybutyrate-co-polyhydroxyvalerate and low density polyethylene. These polymers were filled with flax fibers. Mechanical properties of the composites, biogas production and mass loss under anaerobic digestion have been presented. It has been shown that the mechanical properties sustain after 28 days of biodegradation. Such materials can be found in applications as packaging, as well as in medicine as polymeric scaffolds, and drug delivery systems etc.

Activated carbon’s porous structure allows it to adsorb a substrates, products and catalysts from the environment thus modificated the biocatalysis processes in digestive tract. Active carbons are currently used to remove solvents from gas streams and for water purification; however, few studies have examined the mechanisms of action of active carbon during the biotransformation processes in the digestive tracks. The potential benefits of using activated carbon in feed are uncertain because both its chemical and physical properties can vary significantly depending on the type of carbonaceous feedstock. However, the use of active carbons as dietary supplements can also bring many benefits during biotransformation processes in the gastrointestinal tract. Active carbons can adsorb toxins from the gastrointestinal tract and reduce excessive intestinal gas accumulation. The study concerning the adsorption of bacteria and vitamins on the porous structure of various species of active carbons is an important factor to determine their mechanism of action in biocatalysis in digestive system. The use of properly modified activated carbons as feed additives may have a beneficial effect on the development and functioning of breeding animals in the future. The results of our research show that the active carbon obtained from beech (KB), which contained, on average, 14% oxygen content by weight adsorbed bacteria, such as E. coli and S. aureus, better than all the other active carbons tested. Moreover, the meso- and macropores of carbon seem to contribute little to bacterial adsorption by active carbons. The electron microscopy studies confirmed that the bacteria adhered mainly to the active carbon surface. Our results also indicate that the examined active carbons from beech (KB), coconut shells (TE50), and hard coal (RB2) do not adsorb (or adsorb with very limited efficiency) the vitamins that are routinely added to feed, such as A, B1, D, and K. Broilers fed with feed mixtures supplemented with activated carbon (KB) resulted in increases in the weight of the chickens (∼2%) after 14 days of application and 2% lower feed consumption (conversion) relative to a control sample. Our data indicate that modifying the surface area and elementary content of active carbon may affect its specificity and selectivity and its capacity to absorb particles used in veterinary, human pharmacy, and cosmetology.

Herein, we have investigated the binding interaction of bovine serum albumin (BSA) with a biosurfactant pseudofactin II (PF) in physiological buffer medium through the use of surface tension, fluorescence, circular dichroism (CD), isothermal titration calorimetry (ITC) and molecular docking studies. Fluorescence results showed that PF interacts with BSA through a static quenching mechanism. CD results demonstrate a conformational change in BSA upon PF combination. The negative Gibbs free energies obtained through ITC experiment suggest that the binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process, showing a binding energy of −58.6 kcal mol−1. The present study will help understand the binding mechanism of lipopeptide biosurfactant (PF) to proteins as well as the associated stability and conformational changes.

One hundred and thirty bacterial strains, isolated from Arctic soil on the Svalbard Archipelago, were screened for biosurfactant production. Among them, an isolate identified as Pseudomonas putida BD2 was selected as a potential biosurfactant-producer based on the surface/interfacial activity of the culture supernatant. The ability of the strain to produce simultaneously phosphatidylethanolamines and rhamnolipid, using glucose as a sole carbon source, was demonstrated. The rhamnolipid Rha-Rha-C10-C10 and two homologs of phosphatidylethanolamine were extracted from cell-free supernatant of P. putida BD2 culture with ethyl acetate and identified by UPLC-MS analysis. For Rha-Rha-C10-C10 the surface tension decreased from 72 to 31mN/m and the critical micelle concentration was 0.130mg/mL. The Rha-Rha-C10-C10 was able to form stable aggregates (80-121nm). Pretreatment of a polystyrene surface with 0.5mg/mL rhamnolipid inhibited bacterial adhesion by 43-79% and that of the pathogenic fungal species C. albicans by 89-90%. The same concentration of phosphatidylethanolamines inhibited bacterial adhesion by 23-72% and that of C. albicans by 96-98%. To our knowledge, this is the first report where one type rhamnolipid and two homologs of phospholipid biosurfactants were produced by P. putida isolated from Arctic soil.

It was proposed that magnetic fields (MFs) can influence gene transcription via CTCT motif located in human HSP70 promoter. To check the universality of this mechanism, we estimated the potential role of this motif on plant gene transcription in response to MFs using both bioinformatics and experimental studies. We searched potential promoter sequences (1000 bp upstream) in the potato Solanum tuberosum and thale cress Arabidopsis thaliana genomes for the CTCT sequence. The motif was found, on average, 3.6 and 4.3 times per promoter (148,487 and 134,361 motifs in total) in these two species, respectively; however, the CTCT sequences were not randomly distributed in the promoter regions but were preferentially located near the transcription initiation site and were closely packed. The closer these CTCT sequences to the transcription initiation site, the smaller distance between them in both plants. One can assume that genes with many CTCT motifs in their promoter regions can be potentially regulated by MFs. To check this assumption, we tested the influence of MFs on gene expression in a transgenic potato with three promoters (16R, 20R, and 5UGT) containing from 3 to 12 CTCT sequences and starting expression of β-glucuronidase as a reported gene. The potatoes were exposed to a 50 Hz 60–70 A/m MF for 30 min and the reporter gene activity was measured for up to 24 h. Although other factors induced the reporter gene activity, the MF did not. It implies the CTCT motif does not mediate in response to MF in the tested plant promoters.

The arctic freshwater bacterium Pseudomonas fluorescens BD5 produces biosurfactants when grown on 2% glucose. Crude biosurfactants were extracted from a cell-free culture supernatant with ethyl acetate and purified by preparative reversed phase high performance liquid chromatography (RP-HPLC). The chemical structure of the purified biosurfactants, pseudofactin I and II, was analyzed by matrix assisted laser desorption/ionization time of flight (MALDI TOF) mass spectrometry and tandem mass spectrometry (MS/MS). Both compounds are novel cyclic lipopeptides with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. The C-terminal carboxylic group of the last amino acid (Val or Leu) forms a lactone with the hydroxyl of Thr3. Pseudofactin II reduced the surface tension of water from 72 mN/m to 31.5 mN/m at a concentration of 72 mg/l. Its emulsification activity and stability was greater than that of the synthetic surfactants Tween 20 and Triton X-100; pseudofactins thus have a great potential for application in industrial fields such as bioremediation or biomedicine.

Saccharomyces boulardii is a probiotic strain that confers many benefits to human enterocolopathies and is used against a number of enteric pathogens. Candida albicans is an opportunistic pathogen that causes intestinal infections in immunocompromised patients, and after translocation into the bloodstream, is responsible for serious systemic candidiasis. In this study, we investigated the influence of S. boulardii cells and its culture extract on C. albicans adhesion to Caco-2 and Intestin 407 cell lines. We also tested the proinflammatory IL-1beta, IL-6 and IL-8 cytokine expression by C. albicans-infected Caco-2 cells, using real-time RT-PCR. We found that both S. boulardii and its extract significantly inhibited C. albicans adhesion to epithelial cell lines. The IL-8 gene expression by C. albicans-infected Caco-2 cells was suppressed by the addition of S. boulardii extract. Our results indicate that S. boulardii affects C. albicans adhesion and reduces cytokine-mediated inflammatory host response.

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans. We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans, i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.

Lipopeptide biosurfactants are surface active biomolecules that are produced by a variety of microorganisms. Microbial lipopeptides have gained the interest of microbiologists, chemists and biochemists for their high biodiversity as well as efficient action, low toxicity and good biodegradability in comparison to synthetic counterparts. In this report, we review methods for the production, isolation and screening, purification and structural characterization of microbial lipopeptides. Several techniques are currently available for each step, and we describe the most commonly utilized and recently developed techniques in this review. Investigations on lipopeptide biosurfactants in natural products require efficient isolation techniques for the characterization and evaluation of chemical and biological properties. A combination of chromatographic and spectroscopic techniques offer opportunities for a better characterization of lipopeptide structures, which in turn can lead to the application of lipopeptides in food, pharmaceutical, cosmetics, agricultural and bioremediation industries.

Fatty acid desaturases dehydrogenate acyl chains, which results in the formation of a double bond. Using PCR on flax genomic DNA, we cloned a putative Δ12 fatty acid desaturase (Fad2) gene encoding a 378 amino acid protein. Heterologous expression of this protein in yeast as an N-terminal fusion to GFP showed its localization within endoplasmic reticulum. Analysis of membrane lipids revealed the production of dienoic fatty acids, decreased levels of FAD2 substrates and an increased concentration of longer fatty acids. Higher peroxidation of lipids in FAD2-containing strains is not reflected by any visible phenotype in YPD medium. However, FAD2-containing strains with deleted superoxide dismutase genes exhibited significant growth reductions under oxidative stress.

We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus Candida albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.

Background Flaxseed oil is characterized by high content of essential polyunsaturated fatty acids (PUFA) promoted as a human dietary supplement protecting against atherosclerosis. The disadvantage of the high PUFA content in flax oil is high susceptibility to oxidation, which can result in carcinogenic compound formation. Linola flax cultivar is characterized by high linoleic acid content in comparison to traditional flax cultivars rich in linolenic acid. The changes in fatty acid proportions increase oxidative stability of Linola oil and broaden its use as an edible oil for cooking. However one of investigated transgenic lines has high ALA content making it suitable as omega-3 source. Protection of PUFA oxidation is a critical factor in oil quality. The aim of this study was to investigate the impact of phenylpropanoid contents on the oil properties important during the whole technological process from seed storage to grinding and oil pressing, which may influence health benefits as well as shelf-life, and to establish guidelines for the selection of new cultivars. Methods The composition of oils was determined by chromatographic (GS-FID and LC-PDA-MS) methods. Antioxidant properties of secondary metabolites were analyzed by DPPH method. The stability of oils was investigated: a) during regular storage by measuring acid value peroxide value p-anisidine value malondialdehyde, conjugated dienes and trienes; b) by using accelerated rancidity tests by TBARS reaction; c) by thermoanalytical - differential scanning calorimetry (DSC). Results In one approach, in order to increase oil stability, exogenous substances added are mainly lipid soluble antioxidants from the isoprenoid pathway, such as tocopherol and carotene. The other approach is based on transgenic plant generation that accumulates water soluble compounds. Increased accumulation of phenolic compounds in flax seeds was achieved by three different strategies that modify genes coding for enzymes from the phenylpropanoid pathway. The three types of transgenic flax had different phenylpropanoid profiles detected in oil, highly increasing its stability. Conclusions We found that hydrophilic phenylpropanoids more than lipophilic isoprenoid compounds determine oil stability however they can work synergistically. Among phenolics the caffeic acid was most effective in increasing oil stability.

Three approaches were successfully used to manipulate content of flavonoids in transgenic plants. Overexpressing either the adaptor 14-3-3 protein or genes coding the key enzymes of the flavonoid biosynthesis pathway resulted in a significant increase in the compound content in potato tuber epidermis. The opposite effect was observed in transgenic plants in which these proteins were repressed; this strongly supports the view that the gene construct determines transgenic plant features. The most effective construct was, however, the one containing single dihydroflavonol reductase (DFR) gene in sense orientation. In all cases the increase in flavonoid content resulted in the expected enhancement of the antioxidant capacity of tuber extract. At the biochemical level a decrease in the starch content in transgenic plant overexpressing proteins regulating flavonoid biosynthesis was detected. In the case of glucosyl transferase (GT) gene overexpression, the content of phenolic compounds remained at the control level, however, the antioxidant capacity of tuber extracts significantly decreased. The GT plants grew faster and were more resistant to pathogen attacks, the tuber yield was significantly higher than that of nontransformants. Thus it is speculated that it is the chemical structure and degree of glucosylation of flavonoids rather than their quantity which determines transgenic plant features.

Anthocyanidine glucosyltransferase (GT) is a key upregulating enzyme in anthocyanins stability. A 1.6 kb-GT promoter from Solanum sogarandinum has been isolated and its sequence analysed. The inspection of the promoter sequence revealed several boxes important for the regulation of gene expression. The motifs arrangement was studied using transgenic potato plants transformed with a GT promoter-β-glucuronidase gene fusion construct. The exposure of the transgenic potato to UV, cold, light, as well as the salt and ABA treatment resulted in 5.5-, 6-, 5-, 5- and 8-fold increase in enzyme activity, respectively. The synergistic mode of cold-light and ABA-cold effect has been detected. The UV and cold inducibility of promoter implicates its upregulatory function in flavonoid stability and thus the regulatory role in plant protection against abiotic stresses. In turn, the increase in sucrose concentration (over 2%) in growth medium inhibits GT promoter driven GUS activity. The changes in GT promoter activity upon treatment was confirmed in most cases by western analysis of unmodified plant treated in the same way. The only difference was NaCl treatment that resulted in unchanged GT protein level. Histochemical analysis of GUS under GT promoter revealed the cell specific expression. In leaves the highest expression has been detected in epidermis and spongy mesophyll, but weak staining signals were visible also in other leaf tissues. This pattern became more pronounced in leaves treated with UV. Stem tissues did not show any GUS activity, while in tubers the GUS staining was observed in cortex under periderm and in vascular ring. In roots staining was localized in the root hair zone where rhizodermis and cortex cells are stained.

Consumption of flax (Linum usitatissimum) seeds is beneficial for human health. Flax seeds, containing about 40% of oil, are the richest (among crop plants) source of polyunsaturated fatty acids (PUFA) essential in the human diet. PUFA are highly susceptible to oxidation. Thus only certain cultivars (e.g. Linola) with low linolenic acid content are suitable for commercial preparation of edible oil, which has, nevertheless, a very short shelf life. To study the factors influencing the stability of flax oil, the oil was extracted from nine flax cultivars and analyzed. Linola contained about 3% of linolenic acid while, in other analyzed cultivars, its content ranged from 52% to 73%. Instead, Linola is rich in linoleic acid (about 75%), which in other cultivars varied from 12% to 18%. The susceptibility to oxidation of extracted oil has been analyzed using two methods (measurement of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) formation). Even the low linolenic acid content Linola oil was easily oxidized. The most resistant to peroxidation was the oil extracted from Abby. The potential to reduce peroxidation has been tested using natural antioxidants (β-carotene and quercetin) at concentrations ranging from 10 to 250 μM. The formation of TBARS was most efficiently reduced by 25 μM concentrations of both β-carotene and quercetin. Higher concentrations of β-carotene increased the level of TBARS. The efficiency of β-carotene and quercetin varied, depending on the analyzed cultivar, probably due to intrinsic content of antioxidants.

S. cerevisiae strain delta sodl lacking Cu,Zn-superoxide dismutase and delta sodl delta sod2 mutant lacking both Cu,Zn-SOD and Mn-superoxide dismutase displayed strongly reduced aerobic growth on glucose, glycerol and lactate; delta sod2 deletion had no effect on aerobic growth on glucose and largely precluded growth on glycerol and lactate. The oxygen-induced growth defects and their alleviation by antioxidants depended on growth conditions, in particular on oxygen supply to cells. Under strong aeration, vitamins A and E had a low effect, 100 mumol/L quercetin alleviated the growth defects of all three mutants while beta-carotene had no growth-restoring effect. The superoxide producer paraquat inhibited the aerobic growth of all three mutants in a concentration-dependent manner. Low concentrations of antioxidants had no effect on paraquat toxicity while higher concentrations supported the toxic effect of the agent.

Six full-length cDNAs encoding 14-3-3 proteins from potato (Solanum tuberosum L. cv. Desiree) plants have been recently isolated and sequenced. Screening of a potato genomic library with the 16R cDNA encoding 14-3-3 protein isoform resulted in the identification and isolation of the respective genomic clone. The gene contains four exons and three introns. Inspection of the promoter sequence of the 16R gene revealed several boxes important for the regulation of the gene expression. The induction of the promoter activity by sucrose, IAA, ABA and salicylic acid has been shown. Dof protein-binding sequences, E-boxes and sequences responsible for developmental regulation are most frequently represented. Northern blot and fluorometric analyses, as well as the microscopic examination of transgenic potato plants transformed with GUS reporter under 14-3-3 protein promoter, provide evidence for tissue-specific expression and age-dependent promoter activity. Significant GUS expression was observed in young organs or organ portions, as well as in minor vascular bundles of mature organs.

Poppy seeds are widely used in household and commercial confectionery. The aim of this study was to demonstrate the application of metabolic profiling for industrial monitoring of the molecular changes which occur during minced poppy seed rancidity and brewing processes performed on raw seeds. Both forms of poppy seeds were obtained from a confectionery company. Proton nuclear magnetic resonance (1H NMR) was applied as the analytical method of choice together with multivariate statistical data analysis. Metabolic fingerprinting was applied as a bioprocess control tool to monitor rancidity with the trajectory of change and brewing progressions. Low molecular weight compounds were found to be statistically significant biomarkers of these bioprocesses. Changes in concentrations of chemical compounds were explained relative to the biochemical processes and external conditions. The obtained results provide valuable and comprehensive information to gain a better understanding of the biology of rancidity and brewing processes, while demonstrating the potential for applying NMR spectroscopy combined with multivariate data analysis tools for quality control in food industries involved in the processing of oilseeds. This precious and versatile information gives a better understanding of the biology of these processes.