Application of ESI FT-ICR MS to Study Kraft Lignin Modification by the Exoenzymes of the White Rot Basidiomycete Fungus TrametesHirsutaLE-BIN 072

Trameteshirsuta is a wood rotting fungus that possesses a vast array of lignin degrading enzymes, including7 laccases, 7 ligninolyticmanganese peroxidases, 9 lignin peroxidases and 2 versatile peroxidases. In this study,electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS)was used to examine kraft lignin modification by the enzymatic system of this fungus.The observed pattern of lignin modification suggested that before the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungus started to degrade less oxidized, more recalcitrant, compounds, converting them into the more oxidized forms. The altered pattern of lignin modification enabled changes in the fungal enzymatic system. These changes were further attributed to the appearance of the particular ligninolyticmanganese peroxides enzyme(MnP7), which was added by the fungus to the mixture of enzymes that had already been secreted (VP2 and MnP5). Keywords: wood rotting fungi, kraft lignin, mass spectrometry, peroxidase

Biodegradation Potential of SteccherinumOchraceum: Growth on Different Wood Types and Preliminary Evaluation of Enzymatic Activities

White-rot fungi isa source of a great variety of oxidative and hydrolytic enzymes suitable for biotechnological applications, e.g. in pulp and paper, textile and food industries, bioethanol production, degradation of recalcitrant environmental pollutants,and others. Steccherinumochraceum is a xylotrophicwhite-rot basidiomycetethat can be found in variousclimatic zones on different woody substrates (mostly well decayed). For this research, seventeenstrains of S. ochraceumwere collected in different regions of Russia from various wood substrates (aspen, alder, oak, hazel, birch and willow). Phylogeneticanalyseswere performedbasedon the nucleotide sequences of ITS1, ITS2, 5.8S rRNA, 28S rRNA, β-tubulin and tef1.Oxidaseandcellulaseactivitieswereassessedbyplate-tests with ABTS and CMC. Forevaluation of biodegradation potential,solid state fermentation on alder and pine sawdust wasperformed. Weightanddensitylossaswellas the C:Nratioweremeasuredafter 90 days of cultivation.All S. ochraceum strains exhibited high oxidative activity towards ABTS, indicating secretion of oxidative enzymes (i.e. laccases and class II peroxidases). Cellulase activity was medium or low for most strains and in some strains – absent. Allstrainswereabletodegradealderandpinesawdust. There was no correlation between the enzymatic activity, biodegradation potential and geographic origin of S. ochraceum strains. However, S. ochraceum strains isolated from the same wood substrates exhibited similar characteristics in most cases. Strain LE-BIN 3398 was the most effective for degrading both alder and pine sawdust and could be regarded as a promising source of oxidative enzymes for biotechnology. Keywords: basidiomycetes, biodegradation, solid state fermentation, oxidase activity, Steccherinumochraceu

Purification and Characterization of Two Novel Laccases from Peniophora lycii

Although, currently, more than 100 laccases have been purified from basidiomycete fungi, the majority of these laccases were obtained from fungi of the Polyporales order, and only scarce data are available about the laccases from other fungi. In this article, laccase production by the white-rot basidiomycete fungus Peniophora lycii, belonging to the Russulales order, was investigated. It was shown that, under copper induction, this fungus secreted three different laccase isozymes. Two laccase isozymes—Lac5 and LacA—were purified and their corresponding nucleotide sequences were determined. Both purified laccases were relatively thermostable with periods of half-life at 70 °C of 10 and 8 min for Lac5 and LacA, respectively. The laccases demonstrated the highest activity toward ABTS (97 U·mg−1 for Lac5 and 121 U·mg−1 for LacA at pH 4.5); Lac5 demonstrated the lowest activity toward 2,6-DMP (2.5 U·mg−1 at pH 4.5), while LacA demonstrated this towards gallic acid (1.4 U·mg−1 at pH 4.5). Both Lac5 and LacA were able to efficiently decolorize such dyes as RBBR and Bromcresol Green. Additionally, phylogenetic relationships among laccases of Peniophora spp. were reconstructed, and groups of orthologous genes were determined. Based on these groups, all currently available data about laccases of Peniophora spp. were systematized

In Vitro and In Vivo Antihypertensive Effect of Milk Fermented with Different Strains of Common Starter Lactic Acid Bacteria

Currently, functional dairy products pave a promising way for the prophylaxis of essential hypertension, and the search for new strains capable of producing such products is a constant challenge for scientists around the world. In this study, the antihypertensive properties of milk fermented with several strains of traditional yogurt starters (Lactobacillus delbrueckii strains Lb100 and Lb200; Lactococcus lactis strains dlA, AM1 and MA1; Streptococcus thermophilus strains 159 and 16t) and one strain of non-conventional probiotic starter (Lacticaseibacillus paracasei ABK) were assessed. The in vitro assessment using angiotensin-converting enzyme inhibition assay was performed for all fermentation products, and the best performed products were tested in vivo using Spontaneously Hypertensive Rat (SHR) animal model. In addition, for the best performed products the fatty acid (FA) composition and FA-related nutritional indices were determined. As a result, the milk fermented with two strains (Lb. delbrueckii LB100 and Lc. lactis AM1) demonstrated significant antihypertensive effect during both in vitro and in vivo experiments. Moreover, the milk fermented with Lb. delbrueckii Lb100 demonstrated significantly better FA-related nutritional indexes and lowered total cholesterol in SHRs upon regular consumption. The obtained results can be used in the future to develop new starter cultures producing effective functional antihypertensive dairy products

Whey Protein Hydrolysate and Pumpkin Pectin as Nutraceutical and Prebiotic Components in a Functional Mousse with Antihypertensive and Bifidogenic Properties

Systematical consumption of functional products has a significant positive effect on health and can reduce the risk of diseases. The aim of this study was to investigate the possibility of using whey protein hydrolysate (WPH) and pumpkin pectin as ingredients in a functional mousse, to evaluate the mousse’s antioxidant and hypotensive activities in vitro, and to evaluate the effect of the long-term intake of mousse samples on the progression of hypertension in spontaneously hypertensive rats (SHRs) and on the microbiome status in Wistar rats with antibiotic-induced dysbiosis. The experimental mousse’s in vitro antioxidant activity (oxygen radical absorbance capacity) increased by 1.2 times. The hypotensive (angiotensin-1-converting enzyme inhibitory) activity increased by 6 times in comparison with a commercial mousse. Moreover, the addition of pectin allowed the elimination of the bitter aftertaste of WPH. In vivo testing confirmed the hypotensive properties of the experimental mousse. The systolic blood pressure in SHRs decreased by 18 mmHg and diastolic blood pressure by 12 mmHg. The experimental mousse also showed a pronounced bifidogenic effect. The Bifidobacterium spp. population increased by 3.7 times in rats orally administered with the experimental mousse. The results of these studies confirm that WPH and pumpkin pectin are prospective ingredients for the development of functional mousses

Analytical Characterization of the Widely Consumed Commercialized Fermented Beverages from Russia (Kefir and Ryazhenka) and South Africa (Amasi and Mahewu): Potential Functional Properties and Profiles of Volatile Organic Compounds

In this study, four commercialized indigenous fermented beverages most highly consumed in Russia (kefir and ryazhenka) and South Africa (amasi and mahewu) were analyzed for their potential health-promoting properties and flavor-forming volatile organic compounds (VOC). The analysis of antioxidant capacity demonstrated superiority of dairy-based beverages (kefir, ryazhenka and amasi) over the corn-based mahewu; however, mahewu outperformed dairy-based beverages in terms of its potential antihypertensive effect (i.e., the ability to inhibit angiotensin I converting enzyme). The fatty acid (FA) content of kefir and ryazhenka were more diverse compared to that of amasi, but included a lesser amount of branched chain FA. In terms of calculated FA nutritional indices (e.g., indices of atherogenicity and thrombogenicity), kefir and ryazhenka performed similarly and significantly better than amasi. The agreement between beverages theoretical flavor profiles, which was obtained based on the flavors of individual VOC, and consumers’ flavor perception allow hypothesizing about the contribution of detected VOC to the overall products’ flavor. The obtained data expand current knowledge regarding traditional fermented beverages and their values in terms of national dietary recommendations. Additionally, reported VOC profiles will promote the inclusion of traditional fermented beverages into the rations based on the flavor pairing concept (which is controversial but widely applied)

Comparative Analysis of Peniophora lycii and Trametes hirsuta Exoproteomes Demonstrates “Shades of Gray” in the Concept of White-Rotting Fungi

White-rot basidiomycete fungi are a unique group of organisms that evolved an unprecedented arsenal of extracellular enzymes for an efficient degradation of all components of wood such as cellulose, hemicelluloses and lignin. The exoproteomes of white-rot fungi represent a natural enzymatic toolbox for white biotechnology. Currently, only exoproteomes of a narrow taxonomic group of white-rot fungi—fungi belonging to the Polyporales order—are extensively studied. In this article, two white-rot fungi, Peniophora lycii LE-BIN 2142 from the Russulales order and Trametes hirsuta LE-BIN 072 from the Polyporales order, were compared and contrasted in terms of their enzymatic machinery used for degradation of different types of wood substrates—alder, birch and pine sawdust. Our findings suggested that the studied fungi use extremely different enzymatic systems for the degradation of carbohydrates and lignin. While T. hirsuta LE-BIN 072 behaved as a typical white-rot fungus, P. lycii LE-BIN 2142 demonstrated substantial peculiarities. Instead of using cellulolytic and hemicellulolytic hydrolytic enzymes, P. lycii LE-BIN 2142 primarily relies on oxidative polysaccharide-degrading enzymes such as LPMO and GMC oxidoreductase. Moreover, exoproteomes of P. lycii LE-BIN 2142 completely lacked ligninolytic peroxidases, a well-known marker of white-rot fungi, but instead contained several laccase isozymes and previously uncharacterized FAD-binding domain-containing proteins

Application of ESI FT-ICR MS to Study Kraft Lignin Modification by the Exoenzymes of the White Rot Basidiomycete Fungus TrametesHirsutaLE-BIN 072

Trameteshirsuta is a wood rotting fungus that possesses a vast array of lignin degrading enzymes, including7 laccases, 7 ligninolyticmanganese peroxidases, 9 lignin peroxidases and 2 versatile peroxidases. In this study,electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS)was used to examine kraft lignin modification by the enzymatic system of this fungus.The observed pattern of lignin modification suggested that before the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungal enzymatic system tended to degrade more oxidized moleculesand, hence, less recalcitrant molecules, with the production of hard-to-modify reduced molecular species. At some point after the 6th day of cultivation,the fungus started to degrade less oxidized, more recalcitrant, compounds, converting them into the more oxidized forms. The altered pattern of lignin modification enabled changes in the fungal enzymatic system. These changes were further attributed to the appearance of the particular ligninolyticmanganese peroxides enzyme(MnP7), which was added by the fungus to the mixture of enzymes that had already been secreted (VP2 and MnP5). Keywords: wood rotting fungi, kraft lignin, mass spectrometry, peroxidase

The Fate and Functionality of Alien tRNA Fragments in Culturing Medium and Cells of <i>Escherichia coli</i>

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria

Exoproteome Analysis of Antagonistic Interactions between the Probiotic Bacteria Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F and Multidrug Resistant Strain of Klebsiella pneumonia

The expansion of multiple drug resistant (MDR) strains of Klebsiella pneumoniae presents an immense threat for public health. Annually, this microorganism causes thousands of lethal nosocomial infections worldwide. Currently, it has been shown that certain strains of lactic acid bacteria (LAB) can efficiently inhibit growth of K. pneumoniae and the formation of its biofilms; however, the active principle of such action remains unknown. In the current article, the growth inhibition of MDR K. pneumoniae by two LAB—Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F—is demonstrated, and the nature of this inhibition studied at the level of exoproteome. This article shows that the exoproteomes of studied LAB contains both classically and non-classically secreted proteins. While for L. reuteri LR1 the substantial portion of classically secreted proteins was presented by cell-wall-degrading enzymes, for L. rhamnosus F only one out of four classically secreted proteins was presented by cell-wall hydrolase. Non-classically secreted proteins of both LAB were primarily metabolic enzymes, for some of which a possible moonlighting functioning was proposed. These results contribute to knowledge regarding antagonistic interaction between LAB and pathogenic and opportunistic microorganisms and set new perspectives for the use of LAB to control the spread of these microorganisms