Probiotic Potential and Functional Properties of Lactobacillus Reuteri, Lactobacillus Rhamnosus and Lactobacillus Helveticus: A Comparative Study

This study was conducted to evaluate and comparethe probiotic propertiesofLactobacillus helveticusNK1, Lactobacillus rhamnosusF and Lactobacillus reuteriLR1lactobacilli strains.Changes in pH, cell growth, proteolytic activity, antioxidantactivity, and angiotensin-converting enzyme(ACE)inhibitoryactivity were monitored during fermentation ofreconstituted skim milk (RSM) by pure cultures of lactobacilli.Among the tested strains, L. helveticusNK1 showed the highest proteolytic, ACE inhibitoryand antioxidantactivitiesduring milk fermentation,followed by L. rhamnosus F and L. reuteriLR1.The promising capability of all of the lactobacilli strains to release bioactivepeptides from the milk proteins was demonstrated. Keywords: Lactobacillus, probiotic, milk fermentation, bioactive peptide

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

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

Abstract P-46: Structure of A. Baumannii Phage Tapaz, Revealed with Cryo-Electron Microscopy

Background: Acinetobacter baumannii is an opportunistic pathogen and one of the six most important multidrug resistant microorganisms in hospitals worldwide. Some of its strains are resistant to most of the antibiotics, A. baumannii is included into the Priority 1 part of Global Priority List of Antibiotic-resistant Bacteria. Phage therapy is considered to be an alternative strategy to antibiotic treatments. Methods: A. baumannii strain NIPH601 cells were grown till OD6000.4 and infected with the phage at MOI 10:1. After complete lysis took place cell debris was spined down and phage particles were precipitated with the PEG6000 (final concentration 10% PEG 6000, 0.5 NaCl). Virus particles were collected by centrifugation, resuspended at SM buffer and applied on CsCl step gradient. Gradient was spinned down for 2 hours at 40000g and the fraction containing phage particles was collected and dialyzed against SM buffer. Purified phage particles were applied to Quantifoil 1.2/1.3 grids and plunge-froze in Vitrobot Mark IV (TFS) Micrographs were collected in HKU, Shenzhen campus with Titan Krios cryoelectron microscope (TFS), equipped with Gatan K3 direct electron detector. The micrographs were acquired with 1.06 Å pixel size and 1.5 um average defocus value in counting mode with 50 frames and 1.2 e/Å2/frame dose rate. All image processing was performed with Relion3.0 software, except for the particle picking step performed with cryolo. Results: Lytic A. baumannii phage TaPaz belongs to the family Myoviridae. BLAST search over NCBI “nr” (non-redundant) database revealed close homology with previously published sequences of Acinetobacter phage vB_AbaM_B9 and Acinetobacter phage BS46. However, no structural information about any homologous proteins was found among the PDB structures. The cryo-EM map was reconstructed with single particle analysis independently for the capsid, tail and baseplate regions. The capsid was reconstructed at 3.9 Å resolution with I3 symmetry applied (Fig. 1A). The baseplate region of the phage was reconstructed at 3.5 Å resolution with C3 symmetry (Fig. 1B). The tail region was reconstructed at 2.6 Å resolution with helical symmetry (Rise 36.4 Å, Twist 25.7 deg). Initial atomic model for the tail region was built from sequence with Deeptracer and was further refined in coot (Fig. 1C). Conclusion: We successfully obtained the near-atomic resolution structural map of phage TaPaz. The data obtained contribute to enhancing knowledge of structural diversity of bacterial viruses infecting A. baumannii

Evolutionary Relationships Between the Laccase Genes of Polyporales: Orthology-Based Classification of Laccase Isozymes and Functional Insight From Trametes hirsuta

Laccase is one of the oldest known and intensively studied fungal enzymes capable of oxidizing recalcitrant lignin-resembling phenolic compounds. It is currently well established that fungal genomes almost always contain several non-allelic copies of laccase genes (laccase multigene families); nevertheless, many aspects of laccase multigenicity, for example, their precise biological functions or evolutionary relationships, are mostly unknown. Here, we present a detailed evolutionary analysis of the sensu stricto laccase genes (CAZy – AA1_1) from fungi of the Polyporales order. The conducted analysis provides a better understanding of the Polyporales laccase multigenicity and allows for the systemization of the individual features of different laccase isozymes. In addition, we provide a comparison of the biochemical and catalytic properties of the four laccase isozymes from Trametes hirsuta and suggest their functional diversification within the multigene family

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

Functional Properties and Metabolic Profile of National Fermented Products of Russia and South Africa

. Both Russia and South Africa have a long-standing history of fermented milk product consumption. Along with the products widely distributed around the world, such as yoghurts, in each of these countries there are a number of national products. An example of a widely demanded fermented milk product in Russia is Kefir.This productis used not only as a food source in the diet of children and adults, but also in medical institutions, since ithasa positive effect onhuman health when consumed regularly. South Africa is characterized by the consumption of products such as Amasi,which is produced commercially. Its consumption has also been shown to have beneficial effects on the digestive system. In this research, the metabolic profiles(fatty acid composition and volatile compounds) of these fermented milk products were analyzed and these showed significant differences. The results indicated that this metabolite composition reflected the different production protocols and microbial complexity of these dairy products. The functional properties of the studied drinks were also considered.The average content of L-leucine equivalents in Amasi was slightly higher (6.5-8.9mMol×L −1) than in Kefir (4.9-6.7mMol×L −1). Antioxidant and antihypertensive activity of the fermented products correlated with the depth of hydrolysis of the milk proteins. Amasishowed higher antioxidant and antihypertensive activities (600- 796µМolТE/ml and 1.3-1.5mg/ml, respectively) than Kefir (246-574µМolТE/ml and 2.0-4.3mg/ml, respectively). Keywords: fermented products, Kefir, Amasi,metabolic profile, antioxidant potential, antihypertensive propertie

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

Lignin-degrading peroxidases in white-rot fungus <i>Trametes hirsuta</i> 072. Absolute expression quantification of full multigene family

<div><p>Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete <i>Trametes hirsuta</i> 072, an efficient lignin degrader. The <i>T</i>. <i>hirsuta</i> genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.</p></div