Ruminococcus flavefaciens is among the most important cellulolytic bacterial species in rumen and gastrointestinal tract of monogastric herbivorous animals. Its extraordinary efficiency in degradation of (hemi)cellulosic substrates is associated with the production of remarcably intricate extracellular multienzyme complexes, named cellulosomes. However, the vast majority of information on R. flavefaciens (hemi)cellulolytic system is exclusively based on either gen(om)e sequence analysis or analysis of overexpressed products of cloned genes and therefore reflect the genomic potential rather than actual state of expression. Except for some early studies, little is known about the substrate-driven expression patterns of proteins involved in the degradation of cellulose and their partitioning between the cell surface and supernatant. This information is crucial for understanding the roles and contributions of different proteins in degradation of cellulosic substrates, which is not only important for understanding the natural cellulolytic systems, but also for optimization of industrial processes involved in bioenergy extraction from cellulosic waste. Many genes with cellulosome-related sequences have been found in R. flavefaciens 17 and FD-1. Interestingly, two variants of another R. flavefaciens strain 007 (C and S) are known to differ exclusively in their ability to degrade cotton cellulose, but nothing is known about their cellulosome organization. In the present work we investigated the cellulolytic system of these two mutants at the genomic as well as proteomic level. The draft genome sequence revealed identical organization of sca gene cluster (encoding cellulosomal structural proteins) as has previously been found in four other strains of R. flavefaciens, namely 17, FD-1, C94 and JM1. The cluster consists of five genes in the following order: scaC-scaA-scaB-cttA-scaE. The translated sequences and modular structure of the putative scaffoldins are almost identical to homologues from strain 17. The scaffold of each cellulosome from R. flavefaciens 007 therefore consists of one anchoring scaffoldin ScaE, which binds the largest scaffoldin ScaB. The latter binds up to seven molecules of second largest scaffoldin, ScaA, which can accomodate up to three catalytic subunits directly or indirectly via the adaptor scaffoldin, ScaC. The cellulases of R. flavefaciens 007 belong to four families of glycoside hydrolases, namely GH48, GH44, GH9 in GH5. Majority of these enzymes are putative endoglucanases, belonging to families GH5 (8 genes) and GH9 (14 genes), whereas only one gene encoding GH44 and GH48 was found. Apart from catalytic domains, most of these proteins also contain dockerins – signature sequences, which indicate their attachement to cellulosomes. On the other hand, carbohydrate-binding modules were only coupled to GH9 catalytic domains. Analysis of the extracellular proteomes revealed that the cellulosomes of Avicel-grown cultures are composed of three major catalytic subunits – putative endoglucanase from family GH9, putative exoglucanase from family 48 and putative xylanase/mananase from from family GH26. Apart from the three major enzymes, a wider spectrum of other proteins with endoglucanase activity is also expressed in much lower concentrations. Larger endoglucanases with Mw similar to putative dockerin and/or CBM-containing GH9 or GH44 appeared to be constitutively expressed (regardless of the substrate or growth phase), wheras smaller enzymes with Mw similar to putative free GH5 were only detected in stationary phase of Avicel-grown cultures. We hypothesize, that larger, constitutively expressed enzymes play an important role in the first stages of cellulose degradation (i.e. adhesion and amorphogenesis), whereas smaller enzymes are probably specialized for the degradation of already released cellulose chains or larger cellodextrines. Growth on cellulose (in comparison to cellobiose) resulted in increased
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 261692672Probiotic products comprise different functional foods, dietary supplements and drugs. Among the main challenges related to the quality control of probiotic products there is determination of their viability where strain-specificity of the analysis is needed, and monitoring of the phenotypical and genotypical stability of commercial probiotic strains. The producers should provide the methods for the quantification including sample preparation since the formulation have great impact on the results of the quantification. Only a few laboratories are accredited to date for the analysis of probiotic products. In the light of the EFSA’s recent rejection of several probiotic related claims for food products everybody are aware that stronger evidence of the mechanisms of action of probiotics intended for food and dietary supplements is needed. The challenges of functional studies of probiotics include tracking of individual probiotic strains in complex samples such as intestines, faeces, mucosa, etc. A total genome sequencing is expected to become a routine analysis enabling for example in silico identification of strain specific genes or control of genetic stability. Development of probiotic formulations that enable prolonged survival of probiotics at room temperature is also an important topic. The above mentioned problems and challenges will be demonstrated through presentation of selected results of our own research in probiotic field and experiences of our recently established laboratory for the control of probiotic products.
B.04 Guest lecture
COBISS.SI-ID: 3054984The principles of the laboratory animal nutrition are the same as the principles of the nutrition of the other animals or humans. With the proper diet, the balance in the digestion and metabolism of the energy and nutrients in the body of the animals is achieved. Laboratory animals or their tissues and organs are used as "measuring instruments" in biological experiments and with a balanced diet the reliability and accuracy of the experiments could be achieve. Nutrition of the experimental animals is important through whole life, even before the beginning of the experiment, since it has an influence on the reserve of nutrients and through this on the balance in the metabolism at the time of the experiment.
D.10 Educational activities
COBISS.SI-ID: 3175560The study examined the effect of different vitamin E (vitE) isomers, sweet chestnut wood extract (EX-TAN) and their combination on oxidative stress in vivo and oxidative stability of meat in broilers. At age 21-days seventy male broilers were individually caged and divided into six groups of ten. The negative control group (K-Palm) received feed with 7.5% of palm fat, the positive control group (K-Lan) and all the other groups received feed with 7.5% of linseed oil. The feed of control groups were un-supplemented, while other groups were supplemented with vitE or SCW: 85 IU vitE/kg as RRR-α-tocopherol (ERRR85), 85 IU vitE/kg as all-rac-α-tocopherol (E-DL85), 200 IU vitE/kg as all-rac-α-tocopherol (E-DL200), 3 g EX-TAN/kg (TAN) and 85 IU vitE/kg as all-rac-α-tocopherol plus 3 g EX-TAN/kg (E+TAN). After 25-days of experiment the blood, liver and breast muscle samples were taken for determination of markers of oxidative stress. Oxidative stress was determined by measuring lymphocyte DNA damage, malondialdehyde (MDA) in plasma, liver and breast muscle, by analysing the antioxidant capacity of lipid soluble compounds (ACL), total antioxidant status (TAS) in plasma, and by antioxidant enzyme assays. We measured tocopherol concentrations in plasma, liver and breast muscle. The oxidative stability of meat was measured by MDA in differently treated breast meat. Colour, pH, drip loss, electrical conductivity and fatty acid composition of meat were also measured. Both forms and concentrations of vitE as well as combination of vitE and EXTAN reduced DNA fragmentation and MDA formation, while SCW reduced the DNA damage only. There were no influence on antioxidant enzymes and TAS. The addition of vitamin E in feed increased concentrations of VitE in plasma and both tissues. Vitamin E was not able to prevent all the negative effects of lipid oxidation in vivo independent of concentration or forms and only high concentration of vitE improved the oxidative stability of meat. A combination of vitE and EX-TAN showed improvement in some markers of oxidative stress (tocopherol concentration, oxidative stability of meat) in comparison to their individual addition, what could be attributed to synergistic action of vitE and EXTAN.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 3048584