Title of project : PREIMPLANTATION EMBRYO GROWTH AND DEVELOPMENT: INTEGRATION OF REGULATION MECHANISMS
Principal investigator : Koppel J.
Scientific co-workers : Baran V., Cikos S, Rehak P., Vesela J., Mihalik J., Kokardova M., Hlinka D.
Project is focused on the complex study of regulation mechanisms, controlling growth and development. Following questions are considered: understanding of physiological processes and dynamic control mechanisms in preimplantation embryo, influence of enviromental factors on embryo in conditions in vivo and in vitro; precise informations about preimplantation embryo needs during in vitro culture in the defined simplex media without the cocultivation nor serum; study of mechanisms involved in the regulation of cell proliferation and differentiation with the focus on the role of growth factors and developmentally regulated intracytoplasmic proteins in these processes; detailed characterisation of different nucleolar substructures comparing species differences (mouse, rabbit, cow). Following methods are used:
1. Morphology: Electron and light microscopic immucytochemistry: topological organisation of nucleic acids and mapping the sites of transcriptional activity (incorporation of labelled nucleotides), localisation of nucleolar markers (affinity cytochemistry with the specific antibodies).
2. Physiology: IVM/IVF/IVC of mouse and bovine embryos, preimplantation embryo culture in vitro (defined simplex media - modifications of KSOM), morphological and biochemical analysis of developing embryo, micromanipulations (ICSI - mouse and bovine oocyte).
3. Biochemistry: Study of developmental expression of specific mRNA and proteins (stathmin, protooncogenes) in preimplantation embryo using molecular biology methods (RT-PCR) and immunocytochemistry (specific antibodies on LM and EM level)
The collaboration with leading laboratories abroad (INRA, INSERM - France) is helpful in the project realisation.
The list of recent relevant papers:
Vesela J., Cikos S., Hlinka D., Rehak P., Baran V., Koppel J.: Effects of impaired insulin secretion on mouse oocyte fertilization. Hum. Reprod. 10, 1995, 3233-3236.
Baran V., Vesela J., Rehak P., Koppel J., Flechon J.E.: Immunocytochemical localization of fibrillarin and nucleolin in nucleoli of mouse preimplantation embryos. Mol. Reprod. Dev., 40, 1995, 305-310.
Pampfer S., Vanderheyden I., Vesela J., De Hertogh R.: Neutralization of TNF-I action on cell proliferation in rat blastocysts by antisense oligodeoxyribonucleotides directed against TNF-I p60 receptor. Biol. Reprod. 52, 1995, 1316-1326.
Laurincik J., Hyttel P., Baran V., Schmoll F., Niemann H., Brem G., Schellander K.: Corona radiata density as a noninvasive marker of bovine cumulus-oocyte complexes selected for in vitro embryo production. Theriogenology, 46, 1996, 369-377.
Baran V., Flechon J.-E., Pivko J.: Nucleologenesis in the cleaving bovine embryo. Immunocytochemical aspects. Mol. Reprod. Dev., 44, 1996, 63-70.
Rehák, P., Mihalik J., Vesela J., Cikos S., Baran V., Koppel J.: Effects of impaired maternal insulin secretion on preimplantation embryo development in ICR mice. Phys. Res., 45, 1996, 453-458
Title of project: OPTIMIZATION OF DIGESTIVE AND METABOLIC PROCESSES IN RUMINANTS
Principal investigator: Zelenak I.
Scientific co-workers: Fejes J., Jalc D., Sviatko P., Kisidayova S., Kocisova J., Siroka P., Varadyova Z.
Research results: The effect of Yea-Sacc supplement on the level of rumen fermentation products in dependence on the diet has been investigated in the Rusitec. The results showed that Yea-Sacc supplement increased the digestibility of hemicellulose with diet 1 (80% meadow hay + 20% barley) and diet 2 (50% hay + 50% barley). With diet 3 (65% hay + 20% barley + 15% treated beech sawdust) digestibility of dry matter, NDF, hemicellulose and cellulose were significantly lowered. Microbial proteosynthesis increased with diet 1 and 3. From results it followed that the Yea-Sacc additive significantly influenced some aspects of rumen fermentation in vitro, which appeared to be dependent on the nature of diet used. It has been also investigated the relationship between Yea-Sacc and phenolic acids in rumen fermentation. It was found out that 1 mmol HBA (p-hydroxybenzoic acid) had a significant effect on yeast efficacy. This was manifested in the increased degradability of hay dry matter and enhanced total gas production. SYA (syringic acid) in the same amount combined with yeast had a similar effect on gas production , but hay dry matter degradability was not affected.. The result showed a slight effect of phenolic acids and yeast culture on hay rumen fermentation in vitro.
In the experiment with the wheat straw treatment by means of white - rot fungi were evaluated 11 basidiomycetes for their potential to improve degradation of straw. All species of white - rot fungi were incubated on wheat straw for 30 days at 28o C. The rusults demonstrated increasing crude proteine and ash contents in fungi treated straw. The in vitro dry matter digestibilities were increased in 9 white - rot fungi straws. Compared to untreated straw the NDF and ADF contents were reduced in fungi treated straws. Out of three fractions, hemicellulose, cellulose and lignin, the hemicellulose showed the largest proportionate loss and lignin the smallest one in 7 fungi treated straws. The other 4 fungi showed the largest proportionate loss in the hemicellulose and lignin content. The results of this study suggest, that digestibility enhancement of wheat straw by white - rot fungi is regulated by a complex factors including the degradation of structural carbohydrates and lignin.
From rumen protozoa studies resulted that the small ciliate Entodinium caudatum in optimal two-step freezing protocols shows a high cell survival (P> 60 %). Large rumen entodiniomorphid ciliates cannot be reliably kept in vivo, and cryopreservation was investigated as an alternative approach to storage. Three species of large ciliates (Polyplastron multivesiculatum, Epidinium ecaudatum forma caudatum and Ophryoscolex caudatus forma tricoronatus) were sucsesfully cryopreserved by one-step freezing, with mean recoveries of 43, 51, and 80 %, respectively.
The list of recent relevant papers:
Zelenak, I., Jalc, D., Kmet, V., Siroka, P.: Influence of diet and yeast supplement on in vitro ruminal characteristics. Anim.Feed Sci.Technol., 49, 1994, 211-221.
Jalc, D., Zitnan, R., Nerud, F.: Effect of fungus - treated straw on ruminal fermentation in vitro. Anim. Feed Sci. Technol., 46, 1994, 131-141.
Kisidayova, S.: Two-step freezing of rumen ciliate protozoon Entodinium caudatum. J. Microbiol. Meth., 22, 1995, 185-192.
Fejes, J., Varady, J.: The level of faunation of rumen in relation to some factors of nitrogen metabolism. Physiol. Res., 45, 1996, 117-123.
Kisidayova, S.: The cryopreservation of some large ciliate entodiniomorphid protozoa taken from the rumen. Lett. Appl. Microbiol., 23, 1996,389-392.
Jalc, D., Nerud, F., Erbanova, P., Siroka, P.: Effect of white-rot Basidiomycetes treated wheat straw on rumen fermentation in artificial rumen. Reprod. Nutr. Dev., 36, 1996, 263-270.
Jalc, D., Nerud, F., Zitnan, R., Siroka, P.: The effect of white-rot Basidiomycetes on chemical composition and in vitro digestibility of wheat straw. Folia Microbiol., 41, 1996, 73-75.
Zelenak, I., Jalc, D., Siroka, P.: Effect of yeast culture and phenolic acids on the physiology of rumen fermentation determined in vitro. Physiol. Res., 46, 1997, in press.
Title of project : THE MECHANIMS OF RENAL UREA RETENTION IN RUMINANTS
Principal investigator: Leng L.
Scientific co-workers : Faix S., CSc, Szanyiova M., Kocisova J., Kokardova M., Boldizarova K.
The research results of our team obtained from 1994 on the mechanisms of renal urea retention showed that the renal pelvis of sheep is able to modify the concentration of urine flowing out of Bellini ducts to a considerable range. The long - term administration of dDAVP, a synthetic analogue of vasopressin with a prolongated effect, to young growing lambs resulted in a significantly larger development of kidney medulla than cortex. The enlargement of surface area of the renal pelvis by 29 % in dDAVP treated animals favours an increased renal urea reabsorption and its recycling. Morphometric analysis of the adaptation of sheep kidneys to a low dietary intake showed that the increased tubular reabsoption of urea is associated with the significant reduction of the epithelium thickness in both distal convoluted and thick ascending limb while the epithelium of collecting duct was found to be thicker. Micropuncture collections of tubular fluid from the surface segments of sheep nephron revealed a reabsorption of urea from distal tubules of sheep fed high protein diet while no urea reabsorption from the same nephron segment was detected in sheep fed low protein diet. The intraportal infusion of glucagon to sheep resulted in the reduction of glomerular filtration rate what is a completely reverse effect of this hormone on kidney functions of sheep as it is known from the experimental data obtained in simple stomach laboratory animals or humans.
At this time our research is orientated on the role of V2 receptors of vasopressin in the urea transport in kidney structures and on evaluations of the renal functions in sheep with changed morphological proportions of kidneys after the long-term treatment with dDAVP.
The list of recent relevant papers:
Leng, L., Szanyiova, M., Faix, S., Cirio, A.: Renal response of sheep fed low protein diet to intraportal infusion of arginine and glycine. Comp. Biochem. Physiol. 108A., 1994, 343-347.
Szanyiova , M., Leng, L., Faix, S.: Partition of nitrogenous substances in the urine of sheep on different dietary protein intake. Vet. Res., 26, 1995, 27-31.
Faix, S., Szanyiova , M., Leng, L.: Urine concentrations in the spaces of sheep renal pelvis. Vet. Res., 27, 1996, 193-196.
Title of project: BIOTRANSFORMATION OF TOXIC SUBSTANCES BY RUMEN MICROBIAL ECOSYSTEM AND BY RUMINANT'S ORGANISM
Principal investigator: Javorsky P.
Scientific co-workers: Pristas P., Legath J., Holovska K., Lenartova V.
The main scientific goals of the project is the study of the effect of pesticides using in agriculture and forest preservation on the growth and metabolic activities of the rumen microflora of farm or wild ruminants and biodegradation of these substances by ruminal ecosystem, possible the study of the poisonous effects of their secondary metabolites on the ruminants. The project ivolves also the study the effect of environmental pollutants on the levels of antioxidative and detoxicative enzymes in some tissues with the aim to monitor the long term negative impact of low doses of toxic substances on the organism of ruminants.
The study of cumulation of the toxic substances within the organisms of ruminants and their biotransformation is very important, mainly from the point of view the possible contamination of food chain of people. The knowledges about the toxicological effects of these substances on the metabolism of ruminal bacteria and ruminants will be useful for the study of the cumulation and long term effect of the poisonous substances on the health of ruminants. The values of GST activity and the activities of further antioxidative enzymes in the animals tissues could be used as a biomarkers for the monitoring of environmental pollution. The results achieved during realisation of this project will be beneficial for the cases of the critical intoxication of ruminants connected with break-down situation and the information about the toxicological mechanism could be helpful for the therapy of ruminants.
The list of recent relevant papers:
Legath, J., Neuschl, J., Kacmar, P., Poracova, J., Dudrikova, E., Mlynarcikova, H., Kovac, G., Javorsky, P.: Clinical signs and mechanism of supermethrin intoxication in sheep. Vet. Hum. Toxicol., Vol. 34, No. 5, October 1992, pp. 453-455.
Javorsky P., Rybosova E, Mlynarcikova H, Mayerova Z, Legath J.: Resistance of ruminal microorganisms to supermethrin. Vet. Hum. Toxicol., 35 (1), 1993, 15-19.
Rybosova E., Breyl I., Siroka P., Nadaskay R., Javorsky P.: Resistance of lactogenic microorganisms and mixed ruminal bacteria to pentachlorobiphenyl (Delor 105). Bull. Environ. Cont. Toxicol., 51/5, 1993, 651-656.
Title of project : RESTRICTION AND MODIFICATION SYSTEM OF RUMEN BACTERIA.
Principal investigator: Javorsky P.
Scientific co-worker: Pristas P.
The rumen represents the unique ecosystem with different microorganisms and high potential for natural transfer of genetic materials. To save own genetic identity, the various restriction-modification systems could be expected, within the rumen microorganisms. Recently more restriction endonucleases have been isolated and characterized from ruminal bacteria, more of them, have been isolated in our laboratory. All restriction endonucleases detected in ruminal bacteria to date are isoschizomers. These findings extend the variety of their sources. The further study of restriction-modification system of selected ruminal bacterial species have significant implications for genetic manipulation of these bacteria, especially for introduction, expression and stability of foreign DNA within the rumen bacteria and the whole ruminal ecosystem.
The list of recent relevant papers:
Vanat I., Pristas P., Kutejova E., Judova J., Godány A., P. Javorsky.: SbvI restriction endonuclease from Streptococcus bovis. Lett. Apl. Microbiol., 17, 1993, 297 -299.
Vanat I., Pristas P., Rybosova E., Godanyi A., Javorsky P.: SruI, restriction endonuclease from Selenomonas ruminantium. FEMS Microbiol. Lett., 113, 1993, 129 - 132.
Pristas P., Vanat I., Godany A., Javorsky P.: Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5'-AT/TAAT-3'. Arch. Microbiol., 161, 1994, 439-441.
Pristas P., Vanat I., Javorsky P.: Isolation and characterization of a new restriction endonuclease, Sru30DI, from Selenomonas ruminantium, Gene, 1995, 158, 139-140.
Title of project: AGGREGATING BACTERIA IN MICROBIAL INTERACTION OF ANIMAL DIGESTIVE TRACT
Principal investigator: Kmet, V.
Scientific co-workers: Zatkovic, B., Styriak, I., Vajanyiova D., Kohutova V.
The purpose of the project is to enlarge the current knowledge about properties, mechanism of action and possibilities of practical applications of aggregative lactobacilli and oral streptococci in microbial interactions. Lactobacilli are frequently used as a biopreparations in veterinary medicine and food industry. Oral streptococci are considered as a oral vaccine delivery vectors.
Relatively the new group of the competitive agents in lactobacilli and oral streptococci are so called aggregation proteins, which are causing the aggregation (clumping) and coaggregation of pathogenic bacteria ( Wadstrom et al. 1987, Reid et al. 1988, Kmet et al. 1995). Coaggregation is the creation of the aggregates between genetically different strains (Kolenbrander 1993). Multigeneric coaggregation, described in oral bacteria is occuring in various bacterial contents when between two nonaggregating partners the third aggregating bacterium is developing the net of coaagregation bridge.
New aggregative animal Lactobacillus and oral streptococci strains will be isolated from digestive tract of conventional animals and surface proteins will be investigated together with primary metabolite and the production of bacteriocins. The interaction with bacterial pathogens will be investigate in laboratory conditions.
The selection of oral streptococci as a possible recipients (for recombinant plasmid containing E.coli virulence genes) will be in connection with our previous results ( Marekova et al. 1996) on genetic manipulation with rumen Streptococcus bovis (DNA-ase activity, DNA transformation, electroporation, plasmid streptococcal vectors etc.).
The list of recent relevant papers:
Kmet V., Callegari M.L., Bottazi V., Morelli L.: Aggregation-promoting factor in pig intestinal Lactobacillus strains. Letters in Appl. Microbiol. 21, 1995, 351-353
Marekova M., Kmet V., Javorsky P.: Transformation of Streptococcus bovis protoplasts by plasmid DNA. Letters in Appl. Microbiol., 22, 1996, 159-161
Kmet V., Lucchini F.: Aggregation-promoting factor in human vaginal Lactobacillus strains. FEMS Immunol. Med. Microbiol. 00, 1997, 1-5 (accepted in press)
Title of project: NEONATAL NUTRITION AND FUNCTIONAL ACTIVITY OF THE VENTROMEDIAL HYPOTHALAMUS
Principal investigator: Kuchar S.
Scientific co-workers: Mozes S., Rybosova Z.
Main results:
Correlation of the ventromedial hypothalamic (VMH) activity (total RNA content measured by microdensitometer in isolated single neurons) in intact adult male rats with their body fat content (wet weight of the epididymal and retroperitoneal fat pads) showed a significant negative correlation.
Neonatally undernourished rats, in comparison with controls, showed a significantly increased VMH activity on day 15, 30 and 150, and a significantly decreased body fat content on day 150.
In adult rats treated neonatally with monosodium glutamate a significantly decreased VMH activity and a significantly increased body fat were found. Correlation of these two parameters showed also a significant negative correlation.
The list of recent relevant papers:
Mozes, S., Kuchar, S., Michalov, M., Novakova, V.: Influence of litter size on the RNA content of VMH and LHA in preweaning rats. Phys. Behav. 50:1133-1136,1991.
Kuchar, S., Mozes, S., Rybosova, Z., Novakova, V., Michalov, M.: Influence of early undernutrition in rats on their body fat and RNA content in VMH neurons. Physiol. Behav. 52: 695-698, 1992.
Kuchar, S., Mozes, S., Zhang, W.M., Rybosova, Z.: Body fat and RNA content of VMH neurons in rats. Brain Res. Bull. 32: 211-213, 1993.
Novakova, V., Sterc., J., Kuchar, S., Mozes, S.: Maternal behaviour in septal rat females. Physiol. Res. 42: 351-360, 1993.
Mozes, S., Kuchar, S., Rybosova, Z., Novakova, V.: Milk fat concentration and growth of rat pups. Physiol. Res. 42: 29-33, 1993.
Mozes, S., Kuchar, S., Zhang, W.M., Sterc, J.: A simple device for sectioning fresh rat brain. Brain Res. Bull. 34: 419-421, 1994.
Zhang, W.M., Kuchar, S., Mozes, S.: Body fat and RNA content of the VMH cells in rats neonatally treated with monosodium glutamate. Brain Res. Bull. 35: 383-385,1994.
Novakova, V., Sterc. J., Mozes, S.: Oestrous cycle in septal rat females. Physiol. Res. 44: 105-111, 1995.
Title of project : METHODS OF INDIVIDUAL CELL ISOLATION FROM THE EPITHELIUM OF THE GASTROINTESTINAL TRACT AND THE LIVER FOR MICRODENSITOMETRIC QUANTIFICATION OF THEIR ENZYMATIC ACTIVITY.
Principal investigator : Mozes S.
Scientific co-workers : Kuchar S., Martinkova A., Lenhardt L., Svicky E.
The aim of the project is to elaborate standard methods of separation of individual epithalial cells and hepatocytes from chemically unfixed intestine and liver suitable for quantitative cytophotometry. We suppose that the estimation of enzyme activity in the single cells enables on increase of sensitivity and accuracy of measurement as compared to microbiochemical methods or by methods using tissue sections. We expect its subsequent utilization in further research during the examination of developmental changes of the enzymatic activities in the digestive tract of animals and the examination of the relationships of their changes during the pathological conditions. In addition the method of cell isolation can be used also in wider biological research - in all cases where precise microdensitometric quantification of the single cellular component is necessary.
A simple method enabling the microdensitometric analysis of intestinal alkaline phosphatase activity (Azo - coupling methode modified after Burstone; in Lojda et al.: Enzyme histochemistry - A laboratory manual 1979) in single epithelial cells of rats was performed. The isolated cells were obtained by Weiser`s isolation method (Weiser M.M. J. Bioll Chem. 248,2536 - 2541,1973.) modified as follows : small segments of the rat duodenum were removed, everted and immediately frozen after washing. By a succesive incubation of the tissue segments in sodium citrate buffer pH 7.3 for 5 min. epithelial cell were released into a plastic PBS filled tube and centrifuged for 3 min. in 600 x g. The sediment was diluted with distilled water and the individual cells were layered on glass slides and air dryed. The results suggest that such samples of individual cells are morphologically preserved with precise localization of enzyme reaction in brush border.
The amount of final reaction product as measured cytophotometrically in individual cells increased linearly with incubation time (up to 10 min.) By varying the Naphtol AS - BI concentration it increased linearly up to 0.4 mM; a Km value of 0.15 mM was found. In unfixed cryostat sections the amount of final reaction product increased linearly up to 2.0 mM Naphtol AS - BI concentration and a Km of 1.2mM was calculated.
Title of project: ANTIMICROBIAL ACTIVITY AND PLASMID OCCURRENCE IN THE REPRESENTATIVES OF FACULTATIVELY AND STRICTLY ANAEROBIC MICROFLORA IN THE ANIMAL WASTES AND ITS USE IN THE BIOFERMENTATION PROCESSES
Principal investigator: Laukova A.
The concentration of animal production in some regions in Slovakia leads to the excess of organic wastes. Therefore, the wastes and slurries present serious hygienic - epidemiological and epizootological problems mainly from the following aspect:
The use of breeding technologies without litter, especially in pig - farming, poultry - farming and cattle - breeding, cause a large accumulation of the wastes such as a slurry. The stable dung is recyclized but the slurry presents the source of microorganisms, enteric pathogens including. It means, animal organic wastes are considered to be infected because plenty of germs, such as those eliminated in another way than faeces including, are accumulated in the slurry. Once of the possibilities to solve these problems is slurry treating by the methods of aerobic and anaerobic sludges stabilization; which have sufficient inhibition effect mainly in the thermophilic temperature zones. However, the problems are noted during the stabilization processes in psychrophilic and mesophilic temperature zones. That is, there is no possibility to use the high temperature degree for disinfection. Therefore, the application of the bacteria with the ability to produce substances with a broad antimicrobial effect - bacteriocins and/or lantibiotics, is very perspective solution. Moreover, bacteriocin production was found to be plasmid - encoded. It means, the plasmid detection amoung isolates determined is necessary; that is, the conjugative plasmids transfer was already described in the limited polluted environment.
Preliminary Results
First aim of our study was to screen the total counts of selected Gram - positive microflora (enterococci, staphylococci, streptococci and Listeria sp.) presented in catlle's dung.
Second aim of our study was phenotypical characterization of enterococci and staphylococci i. e. percentage of species identified and their distribution as well as to detect their capability to produce bacteriocin substances towards a broad spectrum of bacteria, Listeria spp. including.Third aim was to select a suitable strain with a broad antimicrobial spectrum for further application experiments.
Quantification of the total bacterial counts from cattle's dung was performed at 25th agricultural areas (farms) located in 15th regions of northern - east Slovakia. Fortyfifth samples were examined. The total counts of enterococci raeched approximately 106 cfu/ml. However, the average counts determined was 103 cfu/ml. Of the 52 selected enterococcal isolates, 27 strains were phenotypicaly characterized and allotted to different species. The most frequent counted species were Enterococcus casseliflavus, Ent. faecium and Ent. faecalis. The amount 102 cfu/ml of streptococci was noted in the samples screened.
The counts of staphylococci ranged from 102 cfu/ml to 107 cfu/ml depending on the region. However, atypical species were found such as Staphylococcus auricularis. The majority of species detected represent coagulase - negative staphylococci. Staphyl. xylosus was isolated predominantly. Another species included Staphyl. capitis, Staphyl. sciuri, Staphyl. lentus and Staphyl. warneri. Only Staphyl. aureus was characterized of coagulase - positive staphylococcal species. Important results were obtained in bacteriocin activity tests. Because 36% of enterococci tested showed antagonistic activity towards closely related species, Listeria spp. including. That is, the possibility of competetive interactions in the microbial ecosystem of dung's samples. Because the most of enterococcal bacteriocin substances have predominant antilisterial effect. Our studies supported it. Moreover, Listeria spp. were detected in the samples tested (106 cfu/ml). In addition, four strains were selected for other studies;it means bactericin isolation and characterization. The best characterized is till this time bacteriocin substance produced by Ent. faecalis V24 strain. This substance is the objective of our next experiments. However, here it's real possibility for its further application in biofermentation processes. The similar findings were obtained by testing antibacterial activity of staphylococci. Amoung them, 32% showed activity towards staphylococcal indicator only. However, the experiments are also in work.
Title of project: IMPROVEMENT OF THE QUALITY, NATURALNESS AND SHELF - LIFE OF FOOD PRODUCTS BY THE USE OF SELECTED BACTERIOCINS FROM LACTIC ACID BACTERIA
EU - COPERNICUS PROJECT
Principal investigator: Laukova A.
Bacteriocins are antimicrobial proteins, protein aggregates or protein complexes produced by several bacteria - esoecially lactic acid bacteria - with antagonistic activities against other, more or lessd closely related, bacterial strains. Bacteriocins produced by lactic acid bacteria, agriculturally, industrially important food grade microorganisms as well as microorganisms from the other sources, could be utilised for improving the quality of several food and feed products. This would be achieved by maintaining and enhancing the quality and nutritional value, by increasing freshness and shelf - life, by protection against pathogens, by replacing undesireble preservatives such as nitrate, nitrite and sulfite, and possible other means. Copernicus - project focusses on the application in fermented and non-fermented food products of some of these bacteriocins. It involves transnational collaboration between five research laboratories from four different countries. The joint efforts of the different participants will lead to quality and safety improvements of a variety of food products.
Results
Ent. faecium CCM 4231 is ruminal, bacteriocin - producing strain with a broad antimicrobial spectrum towards different Gram - positive as well as Gram - negative indicators used.
Enterocin CCM 4231 is heat - stable bacteriocin (the remaining activity after its exposition for 5, 15 and 30 min to boiling water was from 400 AU/ml to 200 AU/ml; after heating for 15, 30 as well as for 1 h, 2 h and 24 h at 35°C, the remaining activity ranged from 800 AU/ml to 400 AU/ml; after exposition of bacteriocin at 65°C up to 1 h, activity was 400 AU/ml and 200 AU/ml; after storage of bc T 4°C as well as at - 20°C for one week, activity determined was 800 AU/ml), sensitive to proteolytic enzymes, which showed activity 6 400 AU/ml and 12 800 AU/ml using the main indicators Ent. malodoratus NCDO 846 and Listeria monocytogenes OHIO. Our bacteriocin was purified and its molecular mass was estimated (5 - 8 kDa). The bc production after the growth of producer strain CCM 4231 in different media was also tested. It was found, that bc production is started after 4 h of the beginning of cultivation (800 AU/ml) and maximum of the bc production was noted after 6 h and 8 h after the cultivation (12 800 AU/ml). The bc production was determined also after 24 h of cultivation with decrease character (6 400 AU/ml). Using milk such as cultivation medium, maximum bc production was detected after 8 h of cultivation 6 400 AU/ml. Regarding optimal pH for the production of our bc, the range from pH 4 to pH 7 was found as the best.We reached good results with preliminary application of our enterocin CCM 4231 (3 200 AU/ml) during cheese making procedure as well as during youghurt fermentation process. The decrease of indicators (L. monocytogenes Ohio and Staphyl. aureus SA1) were determined. The similary, the decrease of ruminal strains in the rumen fluid environment was found after bc addition in this environment (3200 AU/ml).
Our study is continued. However, the further application in agroindustry or veterinary medicine is indicated.