Biodegradability Study of Modified Chitosan Films with Cinnamic Acid and Ellagic Acid in Soil.

Currently, natural polymer materials with bactericidal properties are extremely popular. Unfortunately, although the biopolymer material itself is biodegradable, its enrichment with bactericidal compounds may affect the efficiency of biodegradation by natural soil microflora. Therefore, the primary objective of this study was to evaluate the utility of fungi belonging to the genus Trichoderma in facilitating the degradation of chitosan film modified with cinnamic acid and ellagic acid in the soil environment. Only two strains (T.07 and T.14) used chitosan films as a source of carbon and nitrogen. However, their respiratory activity decreased with the addition of tested phenolic acids, especially cinnamic acid. Addition of Trichoderma isolates to the soil increased oxygen consumption during the biodegradation process compared with native microorganisms, especially after application of the T.07 and T.14 consortium. Isolates T.07 and T.14 showed high lipolytic (55.78 U/h and 62.21 U/h) and chitinase (43.03 U/h and 41.27 U/h) activities. Chitinase activity after incorporation of the materials into the soil was higher for samples enriched with T.07, T.14 and the consortium. The isolates were classified as Trichoderma sp. and Trichoderma koningii. Considering the outcomes derived from our findings, it is our contention that the application of Trichoderma isolates holds promise for expediting the degradation process of chitosan materials containing bactericidal compounds.

Effect of birch tar embedded in polylactide on its biodegradation.

The plasticized film was made of polylactide and birch tar, which was used in a concentration of 1, 5 and 10 % by weight. Tar was added to the polymer to obtain materials with antimicrobial properties. The main purpose of this work is to characterize and biodegradation of this film after the end of its use. Therefore, the following analyzes were performed: enzymatic activity of microorganisms in the presence of polylactide (PLA) film containing birch tar (BT), biodegradation process in compost, barrier changes and structural properties of the film before and after biodegradation and bioaugmentation. Biological oxygen demand BOD21, water vapor permeability (Pv), oxygen permeability (Po), scanning electron microscopy (SEM) and enzymatic activity of microorganisms were assessed. Microorganism strains Bacillus toyonensis AK2 and Bacillus albus AK3 were isolated and identified, which constituted an effective consortium increasing the susceptibility of polylactide polymer material with tar to biodegradation in compost. Analyses with the use of the above-mentioned strains had an impact on the change of physicochemical properties, e.g. the presence of biofilm on the surface of the analyzed films and the reduction of the barrier properties of the film, which translates into the recorded susceptibility to biodegradation of these materials. The analyzed films can be used in the packaging industry, and after use, subjected to intentional biodegradation processes, including bioaugmentation.

The Role of Quorum Sensing Molecules in Bacterial-Plant Interactions.

Quorum sensing (QS) is a system of communication of bacterial cells by means of chemical signals called autoinducers, which modulate the behavior of entire populations of Gram-negative and Gram-positive bacteria. Three classes of signaling molecules have been recognized, Al-1, Al-2, Al-3, whose functions are slightly different. However, the phenomenon of quorum sensing is not only concerned with the interactions between bacteria, but the whole spectrum of interspecies interactions. A growing number of research results confirm the important role of QS molecules in the growth stimulation and defense responses in plants. Although many of the details concerning the signaling metabolites of the rhizosphere microflora and plant host are still unknown, Al-1 compounds should be considered as important components of bacterial-plant interactions, leading to the stimulation of plant growth and the biological control of phytopathogens. The use of class 1 autoinducers in plants to induce beneficial activity may be a practical solution to improve plant productivity under field conditions. In addition, researchers are also interested in tools that offer the possibility of regulating the activity of autoinducers by means of degrading enzymes or specific inhibitors (QSI). Current knowledge of QS and QSI provides an excellent foundation for the application of research to biopreparations in agriculture, containing a consortia of AHL-producing bacteria and QS inhibitors and limiting the growth of phytopathogenic organisms.

Endocrine Disrupting Compounds (Nonylphenol and Bisphenol A)-Sources, Harmfulness and Laccase-Assisted Degradation in the Aquatic Environment.

Environmental pollution with organic substances has become one of the world's major problems. Although pollutants occur in the environment at concentrations ranging from nanograms to micrograms per liter, they can have a detrimental effect on species inhabiting aquatic environments. Endocrine disrupting compounds (EDCs) are a particularly dangerous group because they have estrogenic activity. Among EDCs, the alkylphenols commonly used in households deserve attention, from where they go to sewage treatment plants, and then to water reservoirs. New methods of wastewater treatment and removal of high concentrations of xenoestrogens from the aquatic environment are still being searched for. One promising approach is bioremediation, which uses living organisms such as fungi, bacteria, and plants to produce enzymes capable of breaking down organic pollutants. These enzymes include laccase, produced by white rot fungi. The ability of laccase to directly oxidize phenols and other aromatic compounds has become the focus of attention of researchers from around the world. Recent studies show the enormous potential of laccase application in processes such as detoxification and biodegradation of pollutants in natural and industrial wastes.

The Effect of Cold Plasma Pretreatment on Water-Suspended Herbs Measured in the Content of Bioactive Compounds, Antioxidant Activity, Volatile Compounds and Microbial Count of Final Extracts.

Cold plasma is a new technology of promising potential to use as a part of technological extraction lines constructed to implement green chemistry solutions or simply to reduce resources in solvent-based extraction lines. The present study was undertaken to verify the effect of nitrogen cold plasma pre-treatment conducted for 8 min (20 kHz) on the content of antioxidants, antioxidant activity, the profile of volatile compounds, microbial count, pH and color measured in herb extracts (12 herbs: Echinacea purpurea; Salvia officinalis; Urtica dioica; Polygonum aviculare; Vaccinium myrtillus; Taraxacum officinale; Hypericum perforatum; Achillea millefolium; Sanguisorba officinalis; Leonurus cardiaca; Ballota nigra; Andrographis paniculata) obtained with its usage. The surface morphology of extracted herbs was examined as well. Herbs used for extraction were ground and suspended in water before cold plasma treatment, which is a novel approach not studied before. Most plasma-treated extracts were characterized by a higher content of polyphenols (11 out of 12). Content of flavonoids and anthocyanins increased in four extracts and in the case of anthocyanins was significantly higher in comparison to control (up to 77%). The antioxidant activity measured at least by one method (ABTS, DPPH, FRAP) was also higher in nine plasma-treated solutions. Moreover, plasma decreased total aerobic bacteria, affected the color and increased pH of the extracts. The surface structure of the plant material after the extraction process was significantly damaged, which probably led to a higher extraction yield of bioactive compounds and in consequence to the higher antioxidant activity of extracts obtained with the cold plasma treatment.

Exploring the properties of chitinolytic Bacillus isolates for the pathogens biological control.

Plant fungal diseases generate serious losses in the agriculture. The bacteria producing biologically active substances that inhibit the growth of fungal pathogens can be an alternative to the chemicals. The chitinolytic bacteria were isolated from the rhizosphere of wheat (Triticum aestivum L.) and their physiological properties which may be useful in the promotion of plant growth have been investigated. Their chitinases and antifungal activity were studied. The isolates were also tested for indirect growth-promoting traits such as ammonia production, siderophore production, hydrogen cyanide production, and salicylic acid production. Two chitinolytic strains B3 and B5 were identified as Bacillus subtilis and Bacillus sp., respectively. They produced active chitinases on a medium containing shrimp shell powder. The purified chitinases having the molecular weight of 35-45 kDa inhibited the growth of important plant pathogens such as Alternaria alternata, and Fusarium oxysporum. Additionally, the isolates showed the ability to produce a broad range of biological substances promoting the growth of plants.

Purification, characterization and cloning of a chitinase from Stenotrophomonas rhizophila G22.

In the presented research the extracellular chitinase of Stenotrophomonas rhizophila G22 was biochemically and molecularly characterized. The studied enzyme was purified from a 72-h bacterial culture about 14 times, with a recovery of 63%. The molecular weight of the purified protein was estimated at 50 kDa by SDS-PAGE. The enzyme showed high activity against colloidal chitin. Significantly lower activities were observed with native chitin powder and chitosan. Adsorption of the enzyme to colloidal chitin and to powdered chitin at the level of 75% and 37%, respectively, was observed after 30 min of reaction. Optimum temperature and pH were 37 °C and 5.9, respectively. The enzyme demonstrated higher activity against nitrophenyl-ß d N, N', N″-triacetylchitotriose and approx. 5 times lower activity for 4-nitrophenyl-N, N'-diacetyl-ß-d-chitobiose. The enzyme is an endochitinase, which is confirmed by the K m and V max values determined in the studies. S. rhizophila G22 endochitinase was inhibited in the presence of cysteine-specific inhibitors, which indicates the role of cysteine moieties in the mechanism of catalysis or in stabilisation of the enzyme molecule. Also Ca2+ and Mn2+ ions may stabilise the protein's spatial structure. SDS and ions: Fe2+, Cu2+, Co2+, Zn2+ inhibited the activity of enzyme. A full-length (2109 bp) gene coding chitinase from S. rhizophila G22 was obtained. Four domains typical for glycoside hydrolase family 18 (GH 18) chitinases were identified: catalytic Gly_18, chitin-binding-ChtBD3, type-III fibronectin-FN3 and polycystic kidney disease domain-PKD domain.

The effect of inulin supply to high-fat diet rich in saturated fatty acids on pork quality and profile of sarcoplasmic protein in meat exudate.

The aim of the study was to evaluate the influence of inulin supply to high-fat diet rich in saturated fatty acids (SFA) on pork quality and profile of sarcoplasmic protein in drip loss. At 50 days of age, twenty cross-bred pigs (gilts) were randomly allotted to four groups: the control (C) group fed a standard diet, and three experimental (D1, D2 and D3) groups fed a high-fat diet rich in SFA. Moreover, pigs from the groups D2 and D3 consumed an extra inulin supply (7% of daily feed intake) from 85 to 120 days of age (for 5 weeks) and from 50 to 120 days of age (for 10 weeks) respectively. The addition of inulin to the diet reduced meaty odour and flavour significantly, improved tenderness and overall sensory quality of pork and additionally influenced ultimate pH, L* colour parameter, lactate level and protein content in meat. The diets also affected the profile of sarcoplasmic proteins. Significant effects were observed for the following enzymes-PK/PGI (pyruvate kinase/phosphoglucose isomerase) and ALD (aldolase), which are related to the intensity of post-mortem glycolysis. Presented data indicate that long-term inulin supply to high-fat diet has a positive effect on technological and sensory quality as well as protein profile of pork.

Influence of salts and metal nanoparticles on the activity and thermal stability of a recombinant chitinase from Stenotrophomonas maltophilia N4.

Cells of Escherichia coli Rosetta, containing plasmid pET-28a with sequences of DNA of chitinase from Stenotrophomonas maltophilia N4, were used for the efficient synthesis of recombinant chitinolytic enzyme. The objective of this study was to improve thermal stability of the recombinant chitinase by salts and metal nanoparticles (NP). The studied chitinase was thermolabile and largely lost its activity in the first minutes of storage at 50 and 60 °C. The optimum temperature for colloidal chitin hydrolysis by the enzyme was 50 °C. Application of sodium aurothiomalate hydrate and manganese chloride enhanced the activity of the recombinant enzyme. In general, chitinase activity was higher when silver nanoparticles (Ag-NP) were used, but lower for other NP. The thermal stability of chitinase immobilized on Ag-NP and manganese chloride was significantly higher than that of free chitinase. Chitinase thermal stability after gold and manganese oxide nanoparticle application was higher than that of the control at 50 °C. Platinum nanoparticles had no significant effect on thermostability. The Ag-NP had a smaller diameter (from 2 to 20 nm) than Au-NP (from 5 to 70 nm) and Pt-NP (from 4 to 80 nm). The TEM analysis showed that the used NP had a higher affinity for chitinase than for the synthetic substrate. The type, size, and location of the NP on the enzyme played a major role in the thermal stability of chitinase.

Alkaline and Halophilic Protease Production by Bacillus luteus H11 and Its Potential Industrial Applications.

This paper presents the results of the study on the production of protease by Bacillus luteus H11 isolated from an alkaline soda lime. B. luteus H11 was identified as an alkalohalophilic bacterium, and its extracellular serine endoprotease also showed an extreme alkali- and halotolerance. It was remarkably stable in the presence of NaCl up to 5 M. The enzyme was active in a broad range of pH values and temperatures, with an optimum pH of 10.5 and a temperature of 45 °C. It had a molecular mass of about 37 kDa and showed activity against azocasein and a synthetic substrate for the subtilisin-like protease, N-succinyl-l-phenylalanine-p-nitroanilide. The halo-alkaline protease produced by B. luteus H11 seems to be significant from an industrial perspective because of its tolerance towards high salinity and alkalinity as well as its stability against some organic solvents, surfactants and oxidants. These properties make the protease suitable for applications in food, detergent and pharmaceutical industries, and also in environmental bioremediation.

Sarcoplasmic Protein Profile from Drip Loss in Relation to Pork Quality.

The aim of this research was to correlate the sarcoplasmic protein profile of the natural drip loss of muscle with the technological and sensory quality of pork meat. Material for analysis was taken from 14 PenArLan pigs. Simple analysis of relationships between the test protein and meat quality traits showed that they adopt a range of from -0.58 to 0.72. It has been shown that the significance of glycolysis expressed as level of glycogen and lactic acid may be 86% (coefficient of correlations, CR = 0.93), explained by the amount of certain enzymes. The higher relation has been obtained between studied enzymes and value of pH24 , L* and b* parameters of raw meat (98%; CR = 0.99). By contrast, L* and a* parameters, sour flavor, and tenderness of grilled meat in 79% can be explained based on the content of the enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate mutase (CR = 0.89). The results of the multiple regression showed that amount of lactic acid was related to the quantity of the 3 enzymes: phosphofructokinase, AMP deaminase, phosphorylase b/phosphorylase b kinase (r = 0.88).

Production, characterization, gene cloning, and nematocidal activity of the extracellular protease from Stenotrophomonas maltophilia N4.

A rhizosphere strain of the bacterium Stenotrophomonas maltophilia N4 secretes the serine protease PN4, whose molecular mass is approximately 42 kDa. The optimal temperature for the enzyme activity of the 11-fold purified protein was 50°C and the optimal pH was 10.5. The activity of the enzyme was strongly inhibited by specific serine protease inhibitors, which allowed for its classification as an alkaline serine protease family. Ca(2+) ions stimulated the activity of the protease PN4, while Mg(2+) ions stabilized its activity, and Zn(2+) and Cd(2+) ions strongly inhibited its activity. The enzyme has broad substrate specificity. For example, it is able to hydrolyse casein, keratin, albumin, haemoglobin, and gelatin, as well as the insoluble modified substrates azure keratin and azocoll. The gene that encodes the 1740 bp precursor form of the enzyme (accession number: LC031815) was cloned. We then deduced that its amino acid sequence includes the region of the conserved domain of the S8 family of peptidases as well as the catalytic triad Asp/His/Ser. The bacterial culture fluid as well as the purified protease PN4 demonstrated biocidal activity with regard to the nematodes Caenorhabditis elegans and Panagrellus spp.

Biofilm formation on the surface of polylactide during its biodegradation in different environments.

The research was aimed at determining the abundance and viability of biofilm formed on the surface of polylactide (PLA) during its biodegradation in different environments. It was also aimed at isolating biofilm forming bacteria, determining their hydrolytic activity and taxonomic status. The first step was to evaluate PLA biodegradability in lake water, compost and soil, using OxiTop Control. The next step was to assess the ability of isolated bacteria to form biofilm in the investigated environments and to evaluate the biofilm structure. The results indicate that PLA is sensitive to biodegradation in any environment, particularly in compost. During this process biofilm of high viability was observed on the surface of PLA. Based on the 16S rRNA gene sequence, the biofilm-forming bacteria were classified as the following species: Acidovorax sp. LW9, Chryseobacterium sp. LW2, Aeromonas veronii LW8, Arthrobacter aurescens LG2, Arthrobacter sp. LG12, A. aurescens LG9, Elizabethkingia meningoseptica LK3, A. aurescens LK9, A. aurescens and LK7. The results show that different bacterial species formed biofilm of different abundance and hydrolytic activitiy levels.

Biologically-Induced Precipitation of Minerals in a Medium with Zinc Under Sulfate-Reducing Conditions.

Sulfate-reducing microbial communities were enriched from soils collected in areas with crude-oil exploitation. Cultures were grown in modified Postgate C medium and minimal medium, with ethanol or lactate as an electron donor. The batch cultures were grown with addition of zinc in concentrations of 100-700 mg/l. A lack of increased protein concentration in the solutions compared with the control batch, was noted in cultures containing over 200 mg Zn2+/l. The 16S rRNA method was applied to determine the specific composition of the selected microorganism communities. The analysis indicated the presence of Desulfovibrio spp., Desulfobulbus spp. and Desulfotomaculum spp. in the communities. Diffractometric analysis indicated the presence of biogenic sphalerite in cultures with 100 and 200 mg Zn2+/l and elemental sulfur in cultures with 200 mg Zn2+/l. Other post culture sediments (300-700 mg Zn2+/l) contained only hopeite [Zn3(PO4)2·4H2O] formed abiotically during the experiment, which was confirmed by studies of the activity of sulfate-reducing microbial communities.

Purification, characterization, and gene cloning of a chitinase from Stenotrophomonas maltophilia N4.

The Stenotrophomonas maltophilia synthesises high-activity chitinase in response to chitin or chitosan induction. The enzyme was purified 8.5 fold and subjected to characterisation. The optimum hydrolysis conditions for this enzyme when using colloidal chitin as substrate were pH 5.6 and temperature of 45 °C. The enzyme demonstrated high thermal stability at 45 °C within 2 h. The studied chitinase exhibited high activity towards colloidal chitin, glycol chitin and chitosan, while it did not hydrolyse glycosidic bonds in carboxymethylcellulose. The enzyme exhibited the highest activity, equalling 90 U/ml, towards Nitrophenyl ß-D-N,N',N"-triacetylchitotriose and activity of 37 U/ml towards 4-Nitrophenyl N,N'-diacetyl-ß-D-chitobioside. The K(m) value in the presence of the two former substrates was:1.2 and 3.9 mM, respectively, which classifies the studied enzyme as an endochitinase. Cysteine and 2-mercaptoethanol stimulated to a small degree the activity of the chitinase which may indicate the involvement of cysteine residues in the catalysis mechanism. The full length of the nucleotide sequence of this chitinase gene is 2106 bp, which amounts to 702 amino acids.

Production of ligninolytic enzymes by cultures of white rot fungi.

Some Basidiomycota were chosen for studies of key ligninases synthesis (25°C, 30 days) in modified medium (shaken or not cultures) with added wheat straw. Liquid Czapek medium with straw yielded a higher amount of laccase than peroxidase, ground straw induced enzyme worse than chopped straw. With peroxidase the reverse dependencies were observed. Laccase of Lentinus edodes synthesized two enzyme isoforms (ca 30 and 16 kDa). In T. versicolor culture active laccase protein with highest molecular mass ca 65 kDa was found. P. sajor-caju yielded three different peroxidase isoforms. Ligninase biosynthesis depended on strain, straw fragmentation extent, culture method and growth medium.

Chitinolytic microorganisms and their possible application in environmental protection.

This paper provides a review of the latest research findings on the applications of microbial chitinases to biological control. Microorganisms producing these enzymes can inhibit the growth of many fungal diseases that pose a serious threat to global crop production. Currently, efforts are being made to discover producers of chitinolytic enzymes. The potential exists that natural biofungicides will replace chemical fungicides or will be used to supplement currently used fungicides, which would reduce the negative impact of chemicals on the environment and support the sustainable development of agriculture and forestry.

Characterization and mass spectrometry analysis of aminopeptidase N from Pseudomononas putida Lup.

An intracellular aminopeptidase N synthesized by Pseudomonas putida Lup was purified and characterized. The approx. 150-fold purified enzyme showed highest activity against A-beta-naphthylamide at pH 7.5 and at temperature 40 degrees C and was 100% thermostable for 240 min at 40 degrees C. P putida lup aminopeptidase N is a monomer with molecular mass approx. 99 kDa determined by SDS-PAGE and gel permeation chromatography. The enzyme has broad substrate specificity, but is the most active against protein substrates with N-terminal alanine and arginine. The activity of P. putida Lup aminopeptidase N is strongly inhibited in the presence of specific metallopeptidase inhibitors and is partly recovered in the presence of Zn2+ and Co2+ ions. Co2+, Mg2+ and Ca2+ ions increased the activity of the enzyme. Moreover, the enzyme was inhibited by inhibitors of cysteine enzymes. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to PepN of Pseudomonas putida GB-1.

Production of antifungal chitinase by Aspergillus niger LOCK 62 and its potential role in the biological control.

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6 days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43 kDa. The highest activity was obtained at 40 °C for both crude and purified enzymes. The crude chitinase activity was stable during 180 min incubation at 40 °C, but purified chitinase lost about 25 % of its activity under these conditions. Optimal pH for chitinase activity was pH 6-6.5. The activity of crude and purified enzyme was stabilized by Mg(2+) and Ca(2+) ions, but inhibited by Hg(2+) and Pb(2+) ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.

Identification and characterization of a chitinase of Stenotrophomonas maltophilia, a bacterium that is antagonistic towards fungal phytopathogens.

A rhizosphere strain of Stenotrophomonas maltophilia strain MUJ that is strongly antagonistic towards fungal phytopathogens secretes to the culture medium a single form of active chitinolytic enzyme belonging to family 18 of glycosyl hydrolases. The chitinase was purified by a two-stage procedure embracing fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme determined by SDS-PAGE was approximately 52 kDa. The enzyme demonstrated highest activity at 45°C and pH 6.8. The enzymatic protein showed considerable thermal stability during 2 h incubation at 45°C. The activity of the enzyme was strongly inhibited in the presence of Hg²âº and Cu²âº. By applying mass spectrometry analysis, the peptides derived from the purified chitinase were assigned to amino acid sequences of the type ChiA chitinases synthesized by Stenotrophomonas bacteria. The purified enzyme inhibited the growth of fungal phytopathogens belonging to the genera Fusarium, Rhizoctonia and Alternaria.