The aim of this Master Degree Thesis was to monitor the total number of lactic acid bacteria occurring in grape must during wine production. The study was performed on the red wine grape variety Cabernet Moravia from organic vineyard and on the white wine grape variety Sauvignon from both organic and integrated vineyards. The isolation of pure cultures of lactic acid bacteria from mixed cultures and subsequently their identification by genus and species-specific PCR was also subject of the thesis. The experimental results show that the number of viable cells of lactic acid bacteria is influenced not only by the wine grape variety, whether it is a variety of red or white wine grape, but also by the way of wine growing. The method of wine growing also had an impact on the species representation of lactic acid bacteria in each variety.

Les bactéries lactiques (LAB) ont suscité ces dernières années un intérêt accru en agroalimentaire du fait de leur potentiel probiotique, i.e. des potentiels bénéfices santé associés à leur consommation. Les interactions adhésives entre bactéries et composants alimentaires sont susceptibles de jouer un rôle-clé à la fois sur la protection et la répartition des bactéries au sein de l’aliment, impactant donc leur action probiotique. Les travaux réalisés au cours de cette thèse ont pour objectif une meilleure compréhension de ces interactions, afin d’optimiser la fonctionnalité d’aliments contenant des LAB. En effet, le comportement adhésif de la majorité des LAB, ainsi que l’effet des interactions adhésives sur la structuration de l’aliment, sont encore mal connus. En outre, certaines étapes de fabrication alimentaire, telles que l’atomisation, peuvent être génératrices de stress pour les bactéries et donc partiellement compromettre leur capacité à adhérer. Dans le cas où ces bactéries seraient intégrées au sein d’une matrice adhésive, il est également légitime de s’interroger sur les effets de cette adhésion sur la protection des bactéries vis-à-vis du stress infligé. Une méthode de criblage haut-débit a d’abord été développée dans l’objectif d’évaluer rapidement l’affinité adhésive d’une centaine de souches vis-à-vis d’une gamme de biomolécules d’intérêt. Cette méthode a ensuite été appliquée à une collection de 73 souches LAB et a permis de dégager des caractéristiques communes parmi les souches adhérentes, notamment en termes de spécificité d’adhésion. Deux études (expérimentale et théorique) ont été menées conjointement sur l’impact du stress de cisaillement sur la fonctionnalité et l’intégrité des chaînes bactériennes. Ces études suggèrent que la rupture de chaînes bactériennes induite par un stress mécanique serait un processus protecteur de la fonctionnalité bactérienne. Le modèle construit prédit une régiosélectivité des dommages infligés aux cellules bactériennes en chaînes, dont l’intensité dépendrait également de la longueur de chaîne. Appliqué aux interactions adhésives bactéries-particules dans une matrice alimentaire soumise au cisaillement, le modèle suggère un impact défavorable de cette adhésion sur les dommages infligés aux bactéries, d’autant plus important que les particules sont de grande taille. Ce travail pluridisciplinaire apporte ainsi plusieurs éléments-clé qui seront utiles lors la conception et production d’aliments fonctionnels optimisés par rapport à leur action probiotique. / In the last decade, there has been an increasing interest in the potential health effects associated with consumption of Lactic Acid Bacteria (LAB) in foods. Adhesive interactions between bacteria and food components are likely to play a key role on bacterial probiotic action by modulating both bacterial protection and distribution within food matrices. Research presented in this thesis aims a better understanding of these interactions to help optimizing functional food design. Indeed, the adhesive behavior of most LAB, as well as the impact of adhesive interactions on food structuration, remain mostly unknown. Furthermore, some food manufacturing steps, such as spray-drying, may induce stress on bacteria, which can cause partial loss of bacterial adhesive capacities. In case of bacteria integrated within an adhesive matrix, the effect of adhesive interactions on bacterial protection from stress can also be questioned. A high-throughput screening method was first designed to screen quickly a hundred of strains for their adhesive affinities towards a given range of biomolecules of interest. This method was then applied to a 73-LAB strains collection which allowed identifying common characteristics amongst adhesive strains, especially in terms of adhesion specificity. Two studies (experimental and theoretical) were performed in parallel to determine the impact of shear stress on bacterial functionality and bacterial chains integrity. These studies suggest that the stress-induced breaking-down process of bacterial chains can be thought of as a functionality protective process. The proposed model predicts regioselectivity of damages inflicted to bacterial cells within a chain, which intensity would vary with chain length. When applied to bacteria-spherical component adhesive interactions within a food matrix submitted to shearing, the model suggests an unfavorable impact of adhesion on bacterial cell damages, which would be all the more important than spheres are big. This multidisciplinary research project points out several key findings that may help with designing more efficient food matrices for optimized LAB delivery.

La cryopréservation engendre des dégradations variables de l’activité biologique et des fonctionnalités des bactéries lactiques, notamment chez Lactobacillus delbrueckii subsp. bulgaricus, un starter de l’industrie laitière. Le but de ce travail a été d’identifier les marqueurs cellulaires de cryorésitance et de cryosensibilité afin de mieux comprendre les mécanismes de dégradation sous-jacents et d’améliorer les performances industrielles des bactéries lactiques. La cryopresérvation a ici été considérée comme une combination de deux stress majoritaires : froid et osmotique. Une attention particulière a été portée à l’analyse de la membrane cellulaire, un site majeur de dégradation lié à la congélation, mais également à la paroi cellulaire et aux protéines. De plus, les cellules ont été analysées à différentes échelles d’observation, de la population jusqu’à la cellule unique, afin de quantifier l’hétérogénéité des propriétés cellulaires existant au sein de populations. Dans une première partie de ce travail, des conditions de culture ont été comparées pour identifier deux souches de L. bulgaricus présentant des résistances contrastées vis-à-vis de la congélation. Une analyse génomique comparative des souches a également été menée dans le but de fournir des pistes de compréhension de ces comportements différents. Dans une seconde partie, des propriétés membranaires des cellules ont été évaluées en réponse aux stress froid et osmotique : composition en acides gras, organisation au niveau des chaînes d’acides gras et des têtes phospholipidiques, et fluidité.Leur fluidité membranaire a également été caractérisée à une échelle subcellulaire par microscopie de fluorescence au moyen du rayonnement synchrotron, permettant la quantification des hétérogénéités inter- et intra-cellulaires. Enfin, un développement technique et méthodologique a été entrepris afin de permettre l’analyse de bactéries individuelles en milieu aqueux par spectroscopie infrarouge à transformée de Fourier, et ainsi leur signature biochimique en conditions natives. Ces approches complémentaires et multidisciplinaires ont révélé l’existence de propriétés et d’organisation différentes de la membrane des deux souches de L. bulgaricus. Différents types d’interaction entre les molécules cryoprotectrices du milieu extracellulaire et la membrane des deux souches a été proposé, pouvant être à l’origine des dommages causés à la souche sensible. De plus, une hétérogénéité plus importante au sein de la population sensible a été identifiée, attribuée à des différences en termes de composition biochimique et d’organisation au niveau de la membrane et de la paroi. Finalement, ce travail suggère quelques marqueurs cellulaires d’évaluation de la cryorésistance des bactéries lactiques, et fournit des méthodes de caractérisation de l’hétérogénéité biochimique au sein des populations. Ceux-ci pourraient être appliqués à l’étude de toute autre étape critique du procédé de production des bactéries lactiques, et pourraient être utiles pour aller vers la production de ferments homogènes au niveau de leur résistance. / Cryopreservation leads to variable degradation of the biological activity and functionality among lactic acid bacteria (LAB), particularly Lactobacillus delbrueckii subsp. bulgaricus, a dairy starter of industrial relevance. The aim of this work was to identify cellular markers of cryoresistance or cryosensitivity for better understanding the mechanisms of cell cryoinjury and increasing LAB industrial performances. Cryopreservation was here considered as a combination of cold and osmotic stresses. A particular focus was given to the analysis of the cell membrane, recognised as a primary site of cryoinjury, but also of the cell wall and proteins. Moreover, cells were analysed from the population level down to the single-cell level to quantify the heterogeneity of cell properties within populations. In the first part of this work, bacterial cultivation conditions were compared to identify two L. bulgaricus strains with markedly different cell cryoresistance. Moreover, a comparative genomic analysis of the strains was performed to provide some clues for the explanation of their different behaviours. In the second part of this work, the membrane properties were evaluated in response to the cold and osmotic stresses: fatty acid composition, organisation of fatty acyl and phospholipid headgroups, and fluidity.Subcellular membrane fluidity was also characterised by fluorescence microscopy using synchrotron radiation, enabling the quantification of inter- and intra-cellular heterogeneities. Finally, original methodological and technical developments were undertaken to achieve the analysis of individual bacterial cells in an aqueous environment by Fourier transform infrared (FTIR) spectroscopy, for the analysis of the biochemical signature of cells under native conditions. These complementary multidisciplinary approaches revealed different properties and organisation of the membrane of both L. bulgaricus strains. It was proposed that different types of interaction between cryoprotectants of the extracellular matrix and the membrane of both strains could be at the origin of cryoinjury for the sensitive strain. Moreover, a high population heterogeneity characterised the cryosensitive strain, ascribed to differences in terms of biochemical composition and organisation of the membrane and cell wall. Altogether, this work suggests some cellular markers to evaluate LAB cryoresistance and provides methods to characterize population biochemical heterogeneity. These could be applied to any other stressful step of their production process, and should be useful for future production of homogeneous populations of resistant LAB.

G protein-coupled receptors (GPCRs) are key regulatory proteins of immune cell function inducing signaling in response to extracellular (pathogenic) stimuli. Although unrelated, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 share signaling via Gαi/o proteins and the agonist 3-hydroxydecanoic acid (3HDec). Both receptors are abundantly expressed in monocytes, macrophages and neutrophils but have opposing functions in these innate immune cells. Detailed insights into the molecular mechanisms and signaling components involved in immune cell regulation by GPR84 and HCA3 are still lacking. Here, we report that GPR84-mediated pro-inflammatory signaling depends on coupling to the hematopoietic cell-specific Gα15 protein in human macrophages, while HCA3 exclusively couples to Gαi protein. We show that activated GPR84 induces Gα15-dependent ERK activation, increases intracellular Ca2+ and IP3 levels as well as ROS production. In contrast, HCA3 activation shifts macrophage metabolism to a less glycolytic phenotype, which is associated with anti-inflammatory responses. This is supported by an increased release of anti-inflammatory IL-10 and a decreased secretion of pro-inflammatory IL-1β. In primary human neutrophils, stimulation with HCA3 agonists counteracts the GPR84-induced neutrophil activation. Our analyses reveal that 3HDec acts solely through GPR84 but not HCA3 activation in macrophages. In summary, this study shows that HCA3 mediates hyporesponsiveness in response to metabolites derived from dietary lactic acid bacteria and uncovers that GPR84, which is already targeted in clinical trials, promotes pro-inflammatory signaling via Gα15 protein in macrophages.

Growing consumer awareness of the health benefits associated with fruits and vegetables and demand for easy to prepare products has prompted the development of a wide variety of minimally processed fruits and vegetables. Minimally processed fruits and vegetables are often peeled, cut, or diced which compromise the produces’ natural protective barriers, exposing a nutrient rich medium and providing an ideal environment for the growth of microorganisms, including foodborne pathogens. The germination conditions of sprout vegetables consisting of relatively high temperatures and humidity, low light and abundance of nutrients are also conducive to the proliferation of foodborne pathogens. Recent outbreaks and recalls indicate additional measures are needed to improve food safety and maintain the integrity of the food industry. The objective of this research was to evaluate the efficacy of Lactic Acid Bacteria (LAB) against E. coli O157:H7, L. monocytogenes, and Salmonella spp. on apple slices and alfalfa sprouts and it’s influence on product quality. Apple slices inoculated with E. coli O157:H7, L. monocytogenes, and Salmonella spp. (each at 104 CFU/g) were treated with Lb. plantarum alone and in combination with Pediococcus acidophilus and P. pentosaceus (LPP) (107 CFU/g) while alfalfa seeds were inoculated with L. monocytogenes and Salmonella spp. (each at 101 CFU/g and 103 CFU/g) and treated with LPP (107 CFU/g). The growth of the microorganisms on the apple slices was assessed during five and seven days of storage at 4◦C and 20◦C, respectively. Growth on alfalfa seeds was reported during five days of sprouting at 20◦C. Populations of LAB were maintained between 7.0 log CFU/g and 8.0 log CFU/g throughout storage and sprouting on the sliced apples and alfalfa seeds, respectively. Although LAB had no significant effect on pathogen populations on apple slices during storage at 4°C (p > 0.05), populations were significantly different at 20°C (p < 0.05). Populations of L. monocytogenes in the presence of Lb. plantarum and LPP were 1.84 log CFU/g and 2.84 log CFU/g less than the controls after five days of storage at 20°C (p < 0.05). Populations of E. coli O157:H7 in the presence of Lb. plantarum and LPP were 1.83 log CFU/g and 1.86 log CFU/g less than the control after one and three days of storage, respectively. Finally, populations of Salmonella spp. were 0.86 log CFU/g less than populations in the absence of LPP after three days of storage. LPP had a significant effect on the growth of L. monocytogenes and Salmonella spp. on alfalfa seeds (p < 0.05). After five days of sprouting, populations of L. monocytogenes at an initial concentration of 101 CFU/g and 103 CFU/g on seeds treated with LPP were approximately 4.5 log CFU/g and 1.0 log CFU/g less than the untreated seeds, respectively. Populations of Salmonella spp. at an initial concentration of 101 CFU/g and 103 CFU/g were 1.0 log CFU/g less than the control. Overall, on apple slices the combination of Lb. plantarum with P. acidophilum and P. pentosaceus demonstrated greater efficacy than Lb. plantarum alone and reduction of L. monocytogenes by Lb. plantarum and LPP was greater than Salmonella spp. and E. coli O157:H7 on apple slices and alfalfa seeds, alike. LAB had a minimal effect on the quality of the apple slices and alfalfa seeds. LAB could be an effective strategy in reducing pathogen populations at abusive temperatures and germination conditions without influencing the quality of minimally processed fruit and vegetables.

162 leaves printed on single pages, preliminary pages i- vii and numbered pages 1-152. Includes bibliography and abbreviations. / Digitized at 600 dpi grayscale to pdf format (OCR), using a Bizhub 250 Konica Minolta Scanner. / Thesis (PhD(Agric))--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Maize, forage sorghum, lucerne, oats, barley and triticale are the most common silage crops in South Africa, while tropical grasses like Eragrostis curvula and Digitaria eriantha are ensiled to a lesser extent. The aim of this study was to determine the effect of adding a lactic acid bacterial inoculant to E. curvula, D. eriantha, lucerne, forage sorghum, maize and oat silage. The effect of the addition of a lactic acid bacterial inoculant when ensiling E. curvula on the fermentation dynamics during ensiling and the aerobic stability of the silage was determined. The addition of the lactic acid bacterial inoculant to E. curvula at ensiling resulted in a more rapid lowering in pH and improved preservation. Inoculated silage had a higher lactic acid content, less protein breakdown and a lower butyric acid content compared to that of the control silage. Both silages were stable when exposed to air for five days. Digitaria eriantha was ensiled, with or without the addition of a lactic acid bacterial inoculant containing Lactobacillus plantarum. Streptococcus faecium and Pediococcus acidilactici together with the enzymes, cellulase, hemicellulase and amylase. The addition of the inoculant resulted in a more rapid drop in pH, a higher level of lactic acid, an increase in the number of lactic acid bacteria, less protein breakdown and lower numbers of enterobacteria, clostridial spores, yeast and mould compared to the control silage. Digitaria eriantha hay, control and inoculated silage diets were fed to 24 Merino rams (n = 8 per treatment) to determine intake and digestibility. The intake of diets consisting of 90.9% D. eriantha hay, control silage or inoculated silage, differed significantly (p<0.05) at 1395, 1540 and 1848 g DM/day, respectively. The in vivo organic matter digestibility (glkg) of D. eriantha hay, untreated silage and inoculated silage diets was 561, 546, 574, respectively. The addition of the bacterial inoculant when ensiling D.eriantha resulted in better preservation, improved aerobic stability, as well as a higher in vivo organic matter digestibility and intake of D. eriantha silage. The addition of an inoculant or molasses to lucerne (Medicago sativa), ensiled in laboratory silos was investigated. The addition of the additives resulted in an increased preservation rate as indicated by a more rapid lowering of pH, a faster rate of lactic acid production and less protein breakdown compared to control silage. The inoculant was more effective than the molasses in improving the rate of preservation. The aerobic stability of lucerne silage was not affected by inoculation or the addition of molasses. The addition of an inoculant to wilted big bale lucerne silage was studied. The inoculant improved silage quality as indicated by a lower pH, higher lactic acid content, lower ammonia nitrogen content and lower level of butyric acid in inoculated silage compared to the control lucerne silage. The composition of big round bale lucerne silage differed markedly from that of lucerne ensiled in laboratory silos as the former had a higher pH, ammonia nitrogen, butyric acid and acetic acid content and a lower lactic content. Whole crop forage sorghum cultivar FS2 was harvested at the late bloom (20.7% DM) and soft dough (28.9% DM) stages of maturity and ensiled in laboratory silos with the addition of commercial silage inoculants. At both stages of maturity the inoculants caused a more rapid rate of pH decline and a higher amount of lactic acid production. All the silages were well preserved. Silages of the sorghum ensiled at the late bloom stage with all treatments were stable after 5 days of aerobic exposure, whereas sorghum ensiled at the soft dough stage with the addition of the inoculants deteriorated upon aerobic exposure. It is concluded that addition of lactic acid bacterial inoculants to mature sorghum at ensiling might impair the aerobic stability of the silage. The yield, nutritional value and production potential of silage made from twenty one maize hybrids was compared. It was concluded that maize hybrids did differ in metabolizable energy content, rate of digestion, predicted intake and predicted milk production potential. The content of NDF and ADF did not differ between the maize hybrids used in this study and could therefore not be used to predict nutritional value or production potential. Maize was harvested at the hard dough stage and ensiled with or without the addition of a lactic acid bacterial inoculant in laboratory silos and in 210 litre drums. The adding of the inoculant to maize at ensiling did not result in a more rapid drop in pH and higher levels of lactic acid. The intake and growth of South African Mutton Merino lambs fed inoculated and untreated maize silage diets was determined. The average daily gain of lambs fed a diet consisting of either 60% control or inoculated maize silage over a growth period of 60 days was 239 ± 26 and 255 ± 44 g/day, respectively. Although the laboratory study showed very little effect of adding a lactic acid bacterial inoculant to maize at ensiling, lambs tended to consume more of the inoculated silage. In the second study the effect of the addition of a lactic acid bacterial inoculant with an enzyme to maize at ensiling on the fermentation dynamics during ensiling, aerobic stability of the silage, the intake, milk production and milk composition of Jersey cows fed maize silage diets was determined. The inoculant did not result in a more rapid lowering of the pH or a more rapid lactic acid production compared to untreated maize silage made in laboratory silos. Both the control and inoculated maize silages were well preserved. The addition of the inoculant to maize at ensiling improved the palatability, intake and the aerobic stability of maize silage compared to the untreated control maize silage. Milk production, milk composition, live weight and condition score of Jersey cows was not significantly affected by the addition of the inoculant to maize silage. The effect of the addition of an enzyme containing lactic acid bacterial inoculant to big bale oat (Avena sativa, cv Cederberg) silage on silage composition, silage intake, milk production and milk composition of Jersey cows was determined. The crop was cut at the bloom stage, wilted and ensiled in big round bales. The inoculant, Sil-All, was applied during the baling process on half of the bales. Silages were fed to Jersey cows in an intake and milk production study. Both the control and inoculated oat silages were well preserved. The inoculated oat silage had a lower level of butyric acid than the control oat silage. Cows fed the inoculated oat silage produced more (P=O.05) milk (17.7 kg/day) than cows fed the control oat silage (16.7 kg/day). The addition of a lactic acid bacterial inoculant to big bale oat silage improved silage composition and animal performance. This study clearly showed that the composition of silages made in bunker silos under commercial farm conditions differ largely from that of silages made in small scale laboratory silos. When the effect of silage additives on aerobic stability of silage is determined the evaluation should include studies on large scale bunker silages. Evaluation of silage additives should include intake and animal production studies. / AFRIKAANSE OPSOMMING: Mielies, voersorghum, lusem, hawer, gars en korog word algemeen as kuilvoer gewasse benut terwyl tropiese grasse soos Eragrostis curvula en Digitaria eriantha tot 'n mindere mate ingekuil word. Die doel van hierdie studie was om die invloed van 'n melksuurbakterieseinokulant op E. curvula-, D. eriantha-, lusem-, voersorghum-, mielie- en hawerkuilvoer te bepaal. Die invloed van 'n melksuurbakteriese-inokulant op die fermentasiedinamika en die aerobiese stabiliteit van E. curvula-kuilvoer is bepaal. Die toediening van die melksuurbakterieseinokulant tot E. curvula tydens inkuiling het 'n vinniger tempo van pH daling en beter preservering tot gevolg gehad in vergelyking met kontrole kuilvoer. Inokulant behandelde kuilvoer het 'n hoer melksuurinhoud, minder protei'en afbraak en 'n laer bottersuurinhoud as kontrole kuilvoer gehad. Beide kuilvoere was stabiel tydens blootstelling aan lug vir vyf dae. Digitaria eriantha is ingekuil met ofsonder die toediening van 'n melksuurbakteriese-inokulant wat Lactobacillus plantarum. Streptococcus faecium en Pediococcus acidilactici sowel as die ensieme, sellulase, hemisellulase and amilase bevat het. Die inokulant het 'n vinniger tempo van pH-daling, hoer vlakke van melksuur en melksuurbakterie, minder protei'en afbraak en laer getalle van enterobakterie, klostridiale spore, giste and swamme in vergelyking met die kontrole tot gevolg gehad. Digitaria eriantha hooi, kontrole kuilvoer en gei'nokuleerde kuilvoer diete is aan 24 Merino ramme (n = 8 per behandeling) gevoer vir bepaling van inname en verteerbaarheid. Die inname van diete wat uit 90.9% D. eriantha hooi, kontrole kuilvoer of gei'nokuleerde kuilvoer bestaan het, het betekenisvol (p<0.05) verskil en was 1395, 1540 en 1848 gDM/dag, respektiewelik. Die in vivo organiesemateriaal verteerbaarheid (gIkg) vanD. eriantha hooi, kontrole kuilvoer en geYnokuleerde kuilvoer was 561, 546, 574, respektiewelik. Die toediening van die bakteriese-inokulant tydens inkuiling vanD. eriantha het beter preservering, verbeterde aerobiese stabiliteit asook 'n hoer in vivo organiesemateriaal verteerbaarheid van D. eriantha kuilvoer tot gevolg gehad. Die effek van toediening van 'n melksuurbakteriese-inokulant en van molasse tot lusem (Medicago sativa) ingekuil in laboratoriumsilos is ondersoek. Die inokulant toediening en molasse toediening het die tempo van preservering versnel, die pH het vinniger gedaal, melksuur is teen 'n hoer tempo geproduseer en minder proteYen afbraak het plaasgevind in vergelyking met die kontrole kuilvoer. Die tempo van preservering is meer effektief deur toediening van die inokulant verhoog as deur die toediening van molasse. Die aerobiese stabiliteit van lusernkuilvoer is nie beYnvloed deur die toediening van inokulant ofmolasse nie. Die effek van die toediening van 'n melksuurbakteriese-inokulant tot groot rondebaal lusernkuilvoer is ondersoek. Die inokulant het die kwaliteit van die kuilvoer verbeter en het 'n laer pH, hoer melksuur, laer ammoniak stikstofen laer bottersuurinhoud in rondebaallusernkuilvoer tot gevolg gehad in vergelyking met kontrole kuilvoer. Groot rondebaallusernkuilvoer het grootliks verskil van lusernkuilvoer wat in laboratoriumsilos ingekuil is. Die rondebaal kuilvoer het 'n hoer pH, hoer ammoniak-stikstof-, bottersuur- en asynsuurinhoud en 'n laer melksuurinhoud gehad as laboratorium lusernkuilvoer. Voersorghum kultivar FS2 is op die laat blom (20.7% DM) en op die sagte deeg (28.9% DM) stadium met die byvoeging van melksuurbakteriese-inokulante ingekuil in laboratoriumsilos. Toediening van beide inokulante tot sorghum hetop beide die inkuilstadiums gelei tot 'n vinniger tempo van pHdaling en meer melksuurproduksie. Aile kuilvoere insluitend die kontrole kuilvoer was goed gepreserveer. Kontrole sowel geYnokuleerde sorghumkuilvoer ingekuil op die laat blomstadium was stabiel tydens aerobiese blootstelling vir 5 dae. Sorghumkuilvoer ingekuil op die sagtedeegstadium met die byvoeging van inokulante was onstabiel tydens aerobiese blootstelling. Die toediening van melksuurbakteriese-inokulante tot sorghum wat op die sagtedeegstadium ingekuil word kan aerobiese stabiliteit van die kuilvoer grootliks benadeel. Die opbrengs, voedingswaarde en produksiepotensiaal van kuilvoer gemaak van 21 mielie hibriede is vergelyk. Verskille in metaboliseerbare energie inhoud, tempo van vertering, voorspelde inname en voorspelde melkproduksie het tussen mielie hibriede voorgekom. Die neutraalbestandevesel- en suurbestandeveselinhoud het nie verskil tussen hibriede nie en derhalwe kon dit nie gebruik word om voedingswaarde ofproduksiepotensiaal te beraam rue. Mielies is op die hardedeegstadium met of sonder die toediening van 'n melksuurbakterieseinokulant in laboratoriumsilos en 210 liter dromme ingekui!. Die toediening van die inokulant het geen invloed op tempo van pH-daling ofproduksie van melksuur gehad nie. Die inname en groei van SA Vleismerino lammers wat 'n dieet bestaande uit 60% kontrole of inokulant behandelde mieliekuilvoer ontvang het, is bepaa!. Die gemiddelde daaglikse toename van lammers was 239 ± 26 and 255 ± 44 g/dag vir die kontrole en inokulant mieliekuilvoer dieet respektiewelik. Alhoewel die laboratoriumstudie weinig verskille tussen die kontrole en die geYnokuleerde mieliekuilvoer getoon het, het lammers geneig om meer van die geYnokuleerde mieliekuilvoer in te neem. In die tweede studie met mieliekuilvoer is die effek van toediening van 'n melksuurbakteriese-inokulant met ensieme, op die ferrnentasiedinamika tydens inkuiling, die aerobiese stabiliteit van mieliekuilvoer asook die inname, melkproduksie en melksamestelling van Jersey koeie bepaa!. Die inokulant het nie die tempo van pH daling en produksie van melksuurverhoog nie en beide kontrole en geYnokuleerde mieliekuilvoerwas goed gepreserveer. Die toediening van die inokulant tot mieliekuilvoer het die smaaklikheid, inname en die aerobiese stabiliteit van mieliekuilvoer verhoog in vergeiyking met kontrole mieliekuilvoer. Melkproduksie, melksamestelling, liggaamsmassa en kondisiepunt van Jersey koeie is nie betekenisvol beYnvloed deur die toediening van die inokulant tot mieliekuilvoer nie. Die effek van die toediening van 'n ensiem bevattende melksuurbakteriese-inokulant tot groot rondebaal hawer (Avena sativa, cv Cederberg) kuilvoer op die samestelling van kuilvoer, kuilvoerinname, melkproduksie en melksamestelling van Jersey koeie is bepaa!. Die gewas is gesny op die blomstadium, verwelk en as rondebaalkuilvoer gepreserveer. Die inokulant, Sil-All, is tydens die baalproses toegedien op die helfte van die bale. Kuilvoere is aan Jersey koeie gevoer in 'n inname en melkproduksiestudie. Beide die kontrole en geYnokuleerde hawerkuilvoer was goed gepreserveer. Die bottersuurinhoud van geYnokuleerde hawerkuilvoer was laer as die van die kontrole hawerkuilvoer. Koeie wat geYnokuleerde hawerkuilvoer gevoer is het meer (P=0.05) melk (17.7 kg/dag) geproduseer as koeie wat kontrole hawerkuilvoer ontvang het (16.7 kg/dag). Die toediening van 'n melksuurbakteriese-inokulant het kuilvoer kwaliteit en diereproduksie verbeter. Hierdie studie wys duidelike verskille uit tussen kuilvoer wat in bunkersilos onder kommersiele toestande ingekuil is, en kuilvoer wat in laboratoriumsilos gemaak is. Wanneer die effek van kuilvoerbymiddels op die aerobiese stabiliteit van kuilvoer bepaal word behoort finale evaluasie gedoen te word op kuilvoer gemaak in bunkersilos soos onder plaastoestande plaasvind. Evaluasie van kuilvoerbymiddels behoort inname en diereproduksiestudies in te sluit.

Ce travail de thèse a permis de décrire la contamination importante des grains du café de (Coffea canephora variété robusta) par des moisissures au cours des traitements post récolte des cerises de café par la voie sèche, dans une zone tropicale humide. La stratégie d’échantillonnage mise en place à consister à faire des prélèvements durant deux années successives (2008 et 2009) sur des sites localisés dans les principales zones de production du café en Côte d’Ivoire. A partir de 31 échantillons de cerises, grains et coques de café, 218 souches sauvages de moisissures ont été isolées sur milieu Potato Dextrose Agar (PDA) et identifiées. Ces champignons filamenteux sont répartis comme suit : Aspergillus section Nigri (52%) ; Aspergillus verts (13%), Penicillium (10%), Mucor (16%), Fusarium (4%), autre (5%). Les Aspergillus section Nigri qui comptent le complexe Aspergillus niger agreggate et Aspergillus carbonarius représentent un peu plus de la moitié de la population fongique soit 52%, soit 30% en 2008 et 70% en 2009, de la flore fongique totale. Ce groupe a fait l’objet d’une caractérisation morphologique. L’étude du potentiel mycotoxinogène des Aspergillus section Nigri isolées du café robusta a démontré qu’en plus de l’OTA, certaines souches d’Aspergillus Nigri produiraient de l’aflatoxine. Cependant l’espèce Aspergillus carbonarius reste la plus ochratoxinogène (0,6 µg et 15µg d’OTA/g de milieu gélosé). En plus des moisissures 44 souches de bactéries lactiques (LAB) ont été isolées à partir de la pulpe fraîche de café. Les caractères morphologiques, biochimiques et culturaux ont été étudiés. L’identification moléculaire des bactéries a permis de les classer dans le groupe de Lactobacillus plantarum sp. Après un criblage orienté, deux souches de LAB avec un effet important d’inhibition de croissance des moisissures mycotoxinogènes ont été sélectionnées. Les deux souches de Lactobacillus plantarum ont démontré une activité antifongique contre les souches d’Aspergillus carbonarius hautement ochratoxinogènes. Par conséquent la prévention de la mycotoxinogenèse sur café robusta, pourrait passer par l’inhibition de la croissance de certaines moisissures ochratoxinogènes. Les résultats acquis au cours de ce travail de thèse serviront de base afin de poursuivre cette étude d’une part avec des essais in situ pour tester l’efficacité des LAB sélectionnées et d’autre part, rechercher les biomolécules actives contre la germination des spores contaminants naturels post récolte en particulier des cerises de café en Côte d’Ivoire et des fruits et légumes en général. / One of the objectives of this thesis was to describe the significant contamination of robusta coffee beans (Coffea canephora) by moulds during the post-harvest processing of coffee cherries in the dry process. The sampling strategy was to take samples for two consecutive years (2008 and 2009) from different areas of coffee production in Ivory Coast and on the other hand, from the same area but from coffee producers using different methods of drying of coffee beans. From 31 samples, 218 wild strains of fungi were isolated on Potato Dextrose Agar (PDA) media and identified. These filamentous fungi were as follows: black Aspergilli (52%); green Aspergilli (13%), Penicillium (10%), Mucor (16%), Fusarium (4%) and others (5%). The black Aspergilli were found to include Aspergillus niger and Aspergillus carbonarius representing 52% of the fungal population, with a proportion of 30% in 2008 and 70% in 2009 of the total fungal flora. This group was selected to study more about their mycotoxin production. Most strains grown on media and at specific incubation conditions, were capable of producing one or more kinds of mycotoxins. Analysis of mycotoxins from fungi isolated from less than a hundred robusta coffee showed that ochratoxin A (OTA) was not the only mycotoxin that may contaminate the robusta coffee in Ivory Coast. Indeed, several strains belonging to the species Aspergillus Nigri group had shown their ability to produce not only ochratoxin A but also aflatoxin. However, the species A. carbonarius remains as the most ochratoxigenic strain but it does not produce aflatoxin.In parallel to the isolation of fungi, 44 strains of lactic acid bacteria (LAB) were also isolated from fresh coffee cherries, harvested in Ivory Coast in 2009. The morphological, biochemical and growth characteristics were studied. Molecular identification of strains ranked them to be in the group of Lactobacillus plantarum sp. After a screening experiment, it was possible to select two strains of LAB with a significant effect of inhibiting fungal growth by producing mycotoxins. The two strains of Lactobacillus plantarum showed antifungal activity against strains of Aspergillus carbonarius which is highly ochratoxigenic. Therefore the prevention of mycotoxigenicity of robusta coffee, could be rised by inhibiting the growth of certain ochratoxigenic fungi. The results achieved in this thesis serve as a basis to continue the study on one hand with field trials to test the effectiveness of selected LAB on the other hand, look for active biomolecules against spore germination of contaminants especially the natural post-harvest coffee beans in Ivory Coast and fruits and vegetables in general.

Nesta pesquisa, estudaram-se as variações ocorridas na relação entre autólise de culturas lácticas e o desenvolvimento da proteólise de queijo Prato produzido em quatro regiões brasileiras: Santa Catarina (Queijo A), Goiás (Queijo B), São Paulo (Queijo C) e Minas Gerais (Queijo D). A análise quantitativa da população de bactérias lácticas durante a maturação mostrou perfis microbiológicos similares para todas as amostras de queijos examinadas. Após 5 dias de maturação, lactococos e estreptococos estavam presentes em números mais elevados do que lactobacilos mesofílicos e termofílicos, leuconostoc e fermentadores de lactato. Contudo, essas populações aumentaram consideravelmente no final do processo de maturação. Enterococos e fermentadores de citrato permaneceram em números relativamente reduzidos ao longo da maturação. A análise qualitativa mostrou a predominância de \"non starter lactic acid bactéria\" (NSLAB) nos queijos das quatro origens, principalmente de Lactobacillus sp. Outros gêneros foram identificados em menor proporção: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. e Streptococcus sp. O queijo C diferiu dos demais por não apresentar Pediococcus sp. e Streptococcus sp. As culturas lácticas adicionadas Lactococcus lacfis sp. e Leuconostoc sp. estavam presentes em populações menores. A autólise foi estudada pela determinação da atividade de aminopeptidases e detecção de autolisinas por zimogramas e de enzimas intracelulares por \"imunoblotting\". Uma maior liberação de aminopeptidases ocorreu no queijo D, seguido dos queijos C, B e A. Não foram detectadas bandas de atividade lítica nos zimogramas dos queijos A e B em todas as condições avaliadas. Nos zimogramas, detectou-se uma banda de 30 KDa nos queijos C e D a pH 7,4 e 44&#176;C, e uma outra de 40 KDa, exclusiva no queijo D, ambas de fraca intensidade. A pH 6,8 e 42&#176;C, detectou-se bandas de 40KDa de fraca intensidade no queijo C e forte no D, além de mais duas de fraca intensidade de 90KDa e 110KDa no queijo D. Na análise em \"imunnoblotting\" com o antisoro anti-Lc, foi observado apenas sinais fracos de reação positiva e em números inferiores àquelas reveladas com o citoplasma bruto de Lac. Lacfis subsp. lacfis (controle positivo), indicando que a autólise foi praticamente inexistente. Com o antisoro anti-D-LDH, também não se detectou sinais de reação positiva nos queijos A e B, enquanto nos queijos C e D, verificou-se sinais positivos de 37KDa, de forte intensidade e correspondentes à proteína D-LDH, indicando a lise de Lab. helveticus. A evolução da proteólise foi determinada quantitativamente durante a maturação e avaliada com base nos índices: NS-pH4,6/NT% e NNP/NT%, teor de tirosina, eletroforese (Uréia-PAGE) e quantificação de aminoácidos individuais livres. Não foram detectadas diferenças significativas entre os queijos A. B, C e D no início da maturação. Contudo, com a fragmentação das proteínas, ocorreu um aumento gradual desses índices, tendo-se observado valores mais elevados no queijo D, seguido dos queijos C, B e A. Os perfis eletroforéticos de proteínas foram similares para os queijos das quatro origens e mostraram claramente que o coagulante e a plasmina foram os responsáveis pela degradação inicial das caseínas. A taxa de degradação da &#945;s1- e &#946;-caseína apresentou a seguinte ordem: D &gt; C &#8805; B &gt; A. O acúmulo de aminoácidos livres também foi mais rápido no queijo D, seguido dos queijos C, B e A. Portanto, a autólise de Lab. helveticus no queijo D acelerou a proteólise, diminuindo o período de maturação em 45% e não afetando negativamente o desenvolvimento de \"flavour\" e nem a textura. No final da maturação (45 dias), os compostos voláteis foram determinados por meio de cromatografia gasosa com espectrometria de massa (CG-MS). Com raras exceções, os queijos das quatro origens continham os mesmos compostos voláteis, embora em quantidades distintas. Os álcoois e ésters foram os compostos majoritários nos queijos A e B e benzaldeído, 3-metil-butanal-2 e hexanal nos C e D. O perfil de textura instrumental (TPA) e a análise sensorial descritiva e quantitativa foram realizados. Os queijos B, C e D apresentaram características mais típicas de queijo Prato, independentemente do fato de que o aroma de manteiga e o sabor doce serem mais acentuados no queijo D. O queijo A foi classificado como tendo as menores características de queijo Prato e apresentou maior nível de defeitos de \"flavour\", principalmente residual e amargor. Os queijos avaliados não apresentaram diferenças significativas quanto à elasticidade e coesividade. Pequenas alterações na composição físico-química dos queijos, principalmente os teores de umidade e de caseína, influenciaram nos parâmetros como a firmeza e a adesividade. O presente estudo demonstrou pela primeira vez a ausência de autólise de Lc. Lacfis sp. em queijo Prato de quatro origens, bem como a ocorrência de autólise de Lab. helveticus nos queijos de duas origens, C e D. A pronunciada autólise dessa espécie teve um impacto positivo na proteólise e foi a responsável pelo aumento da concentração de aminoácidos livres nesses queijos. As diferenças na evolução da proteólise observada entre os queijos C e D, com taxas mais baixas no queijo C, independentemente da autólise pronunciada de Lab. helveticus, foram atribuídas à falta de uniformidade na composição físico-química dos queijos, principalmente pH e os teores de sal na umidade (S/U). / This paper reports a study aimed at evaluating the variations that occur in the interrelationship between autolysis of lactic starter bacteria and the development of proteolysis in Prato cheese produced in four different regions of Brazil: Santa Catarina (Cheese A), Goiás (Cheese B), São Paulo (Cheese C) and Minas Gerais (Cheese D). Quantitative analysis of microbial population yielded similar microbiological profiles for all the cheese samples investigated. After 5 days ripening, lactococci and streptococci were present in higher numbers than mesophilic and thermophilic lactobacilli, leuconostoc and lactate fermenting bacteria. However, the populations of the latter species had considerably increased by the time the ripening process completed 45 days. Enterococci and citrate fermenting bacteria remained present in relatively low numbers throughout ripening. The findings from qualitative analysis confirmed the predominance of non-starter lactic acid bacteria (NSLAB) in the cheeses from four different origins, especially Lactobacillus sp. Other genera of non-starter lactic acid bacteria (NSLAB) were identified in smaller proportions: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. and Streptococcus sp. Cheese C differed from the cheeses in that it no evidence was found of the presence of Pediococcus sp. and Streptococcus sp. The bacteria of the lactic starter culture Lactococcus lactis sp. and Leuconostoc sp. were also found to be present, although in lower numbers. Autolysis was studied by: (1) determination of aminopeptidase activity; (2) detection of autolysins by zymograms and (3) detection of intracellular enzymes by immunoblotting. The release of aminopeptidase was highest in cheese D, followed by C, B and A. No bands of lytic activity were appeared in the zymograms of Cheeses A and B in all conditions evaluated. At pH 7,4 and 44&#176;C, a low-intensity band of 30 KDa was found in cheeses C e D, whereas another low-intensity band was observed only in cheese D. At pH 6,8 and 42&#176;C, bands of 40KDa were observed in cheese C (low intensity) and cheese D (high intensity), in addition to two more low-intensity bands of 90KDa and 110KDa in cheese D. Immunoblotting with antiserum anti-Lc produced only minor signs of positive reaction, evidenced by the formation of low-intensity bands of 100 Kda in cheeses A and B and two high-intensity bands of 75 Kda and 100 Kda in cheeses C and D. Since these were present in smaller numbers to those revealed with crude cytoplasm of Lac. lactis subsp. lactis, it was concluded that autolysis did practically non occur. Immunoblotting with antiserum anti-D-LDH also detected sings of positive reaction in cheeses A and B, whereas in cheeses C e D positive high-intensity signs of 37Kda were found relative to D-LDH protein, indicating lysis of Lab. helveticus. The evolution of proteolysis was determined quantitatively during the ripening process and evaluated on the basis of the following parameters: NS-pH4,6/NT% and NNP/NT% indexes, tyrosine content, electrophoresis (Urea-PAGE) and quantification of free amino acids. No significant differences were found between cheeses A, B, C and D in the ear1y stages of ripening. However, with the on-going fragmentation of proteins during ripening, a gradual increase of the ripening indexes occurred, with the highest values being observed in cheese D, followed by C, B e A. The electrophoretic profiles were similar for the four cheeses investigated and clear1y showed that the clotting agent or milk coagulant and plasmin were responsible for the initial breakdown of the caseins. The degradation rate of Q.sl- and p-casein followed the following order: D &gt; C &#8805; B &gt; A. The buildup of free amino acids was also faster in cheese D, followed by cheeses C, B e A. At the end of the ripening process studied (45 days), the volatile compounds were identified using gas chromatography and mass spectrometry (GC-MS), whereas the instrumental texture profile was measured and evaluated by Texture Profile Analysis (TPA). Cheese samples were evaluated by descriptive and quantitative sensory analysis. With rare exceptions, the cheeses of four different origins contained the same volatile compounds, although in different quantities. Alcohols and esters were the predominant volatile compounds in cheeses A and B and benzaldehyde, 3-methyl-butanal-2 and hexanal in cheeses C and D. Autolysis of Lb. helveticus accelerated proteolysis in cheese D, thereby reducing ripening time by 45% without any negative effect on either flavor or texture development. Cheeses B, C and D exhibited the most typical Prato cheese characteristics, in spite of the fact that the buttery aroma and sweet taste were more pronounced in cheese D. Cheese A was rated as the cheese with the less typical overall Prato cheese profile and was also the one that exhibited the highest degree and number of flavor defects, notably aftertaste and bitterness. The cheeses investigated did not present any significant differences as to elasticity and cohesiveness. Minor changes in the physical-chemical composition of the cheeses - mainly related to the moisture and casein levels - influenced parameters such as firmness and adhesiveness. The present study demonstrates for the very first time the absence of autolysis of Lc. Lactis sp. in Prato cheese from four different origins, as well as the occurrence of autolysis of Lb. helveticus in two of the cheeses analyzed (cheeses C and D). The pronounced autolysis of this species had a positive impact on proteolysis and was responsible for the release of increased quantities of free amino acids in these cheeses. The differences in the evolution of proteolysis observed between cheeses C and D - lower rate of proteolysis in cheese C, in spite of pronounced autolysis of Lb. helveticus - were attributed to poor uniformity of the physical-chemical composition of this cheese, particularly as related to pH and the salt and moisture levels (S/M).

A produção de etanol nas destilarias brasileiras é baseada na atividade fermentativa da levedura Saccharomyces cerevisiae que utiliza o caldo da cana-de-açúcar e/ou o melaço como substrato. Bactérias contaminantes da fermentação alcoólica competem com as leveduras pelos açúcares, afetando o rendimento do sistema produtivo e, consequentemente, causando perdas econômicas significativas às usinas. Biofilmes formados nos tanques de fermentação alcoólica agem como reservatórios de bactérias, contribuindo para contaminações persistentes e de difícil controle. Os biofilmes proporcionam aos seus habitantes certo grau de proteção contra diversas ameaças do meio, incluindo a ação dos antibióticos. Desta forma, o conhecimento da comunidade bacteriana dos biofilmes é fundamental para as medidas que visam o controle das contaminações na produção do bioetanol. No primeiro estudo, a composição e dinâmica da comunidade bacteriana foram determinadas pela análise de sequências do gene 16S rRNA de biofilmes com diferentes períodos de crescimento, correspondentes aos estágios iniciais de estabelecimento destes biofilmes dentro dos tanques de fermentação alcóolica Os resultados mostraram que estas comunidades foram compostas predominantemente pelas bactérias ácido-lácticas (LAB), com destaque para o gênero Lactobacillus. A visualização da estrutura dos biofilmes por microscopia eletrônica de varredura evidenciou que estes são formados por bactérias e leveduras (biofilmes mistos). No segundo estudo, a suscetibilidade aos antimicrobianos (monensina, virginiamicina e beta-ácido derivado do lúpulo) e a capacidade de formação de biofilmes em culturas puras foram avaliadas para isolados de Lactobacillus spp. provenientes de biofilmes (células sésseis) e de vinho bruto (células planctônicas) coletados dos tanques de fermentação. A partir dos resultados foi possível observar que as diferenças na suscetibilidade aos antimicrobianos e na habilidade de formar biofilmes foram estirpe-dependentes e que, em alguns casos, o perfil apresentado para algumas espécies mostrou-se relacionado à fonte de isolamento. Este foi o primeiro estudo sobre biofilmes contaminantes da fermentação alcoólica, em escala industrial, para a produção de etanol a partir da cana-de-açúcar / Bioethanol production in Brazilian distilleries is based on fermentative activity of the yeast Saccharomyces cerevisiae which uses sugarcane juice and/or molasses as a substrate. Bacterial contaminants of alcoholic fermentation compete with yeasts for sugars, affecting ethanol yield and consequently causing relevant economic losses to the fuel ethanol industry. Biofilms formed into fermentors act as bacterial reservoirs, contributing to persistent contaminations that are difficult to control. Biofilms provide a certain degree of protection for their inhabitants against some environmental threats, including antibiotics. Thus, understanding bacterial community within biofilms is essential for actions to control contaminations in bioethanol production. In the first study, composition and dynamic of bacterial community were determined by 16S rRNA gene sequences analysis of biofilms with different growth periods, corresponding to initial stages of biofilm establishment in fermentation tanks. Results showed that these communities were dominated by lactic acid bacteria (LAB), mainly of the genus Lactobacillus. Visualization of biofilm structure by scanning electron microscopy revealed a mixed-species biofilm composed by bacteria and yeasts. In the second study, susceptibility to antimicrobials (monensina, virginiamicina and beta-acids from hops) and capacity to form biofilm in pure culture were evaluated for Lactobacillus spp. isolated from biofilms (sessile cells) and wine (planktonic cells) collected from fermentors. The results showed that differences in the susceptibility to antimicrobials and the ability to form biofilms were strain-specific and, in certain cases, the response of some species was related to the isolation source. This was the first investigation of contaminant biofilms from sugarcane-based alcoholic fermentation on an industrial scale

<p>Cilj rada je bio ispitivanje funkcionalnih karakteristika kombuha napitka<br />od melise (<em>Melissa officinalis</em> L.). Antibakterijska aktivnost kombuha<br />napitaka optimalne konzumne i većih kiselosti ispitana je prema<br />bakterijama izolovanim iz hrane i vode za piće. Nosilac antimikrobne<br />aktivnosti je sirćetna kiselina, a na ostale nosioce ukazuje delovanje<br />neutralisane kombuhe i čajnog napitka prema nekim test bakterijama.<br />Spektrofotometrijskom metodom određen je sadržaj ukupnih fenolnih<br />jedinjenja, a HPLC analizom određen je kvalitativni i kvantitativni sastav<br />fenolnih jedinjenja u fermentacionim tečnostima, čajnim i kombuha<br />napicima od melise i crnog čaja. Antioksidativna aktivnost istih uzoraka<br />ispitana je na DPPH i OH radikale ESR spektralnom metodom. Uzorci<br />fermentacione tečnosti i kombuha napitka od melise imali su veću<br />antioksidativnu aktivnost prema oba radikala u odnosu na uzorke sa<br />crnim čajem. Konzumna kombuha od melise imala je veću<br />antioksidativnu aktivnost od čajnog napitka. Aktivne komponente<br />kombuha napitka od melise su verovatno ruzmarinska kiselina i<br />kvercetin. U ispitivanju antiproliferativne aktivnosti konzumnog<br />kombuha napitka i čajnog napitka od melise na tri ćelijske linije humanih<br />karcinoma: HeLa (epitelni karcinom grlića materice), MCF-7<br />(adenokarcinom dojke) i HT-29 (adenokarcinom debelog creva) utvrđeno<br />je da nije do&scaron;lo do stimulacije proliferacije ispitanih ćelijskih linija pri<br />koncentracijama većim od 100 &mu;g/ml. Pored istraživanja biolo&scaron;ke<br />aktivnosti ispitana je mogućnost simultane mlečno-kiselinske i kombuha<br />fermentacije. Dodatkom starter kultura i <em>Lactobacillus spp</em>. izolata u<br />fermentacionu tečnost dolazi do povećanja sadržaja L- i D- mlečne<br />kiseline, iako su ćelije bakterija mlečne kiseline, osim izolata iz kiselog<br />testa (<em>L. hilgardii</em>), pokazale malu otpornost na uslove tokom<br />fermentacije i čuvanja pripremljenih napitaka. Izvr&scaron;ena je i identifikacija<br />bakterija sirćetnog vrenja izolovanih iz lokalnih čajnih gljiva PCR<br />metodom. Dva izolata verovatno pripadaju vrsti <em>Gluconobacter oxydans</em>,<br />a treći vrsti <em>Gluconacetobacter hansenii</em>.</p> / <p>The aim of this study was to investigate functional characteristics of akombucha beverage from lemon balm (<em>Melissa officinalis</em> L.) tea. Antibacterial activity of kombucha beverages with optimum and higheracidities was tested against bacteria isolated from food and drinking water. The main active component of antibacterial activity was acetic acid, while slight activity of neutralized kombucha and unfermented tea against some test bacteria indicated presence of other antibacterial components. Total phenol concentration in unfermented tea samples, fermentation broths and kombucha beverages from lemon balm and black tea was determined spectrophotometrically whereas qualitative and quantitative concentration of polyphenolic compounds was determined by HPLC method. Antioxidant activity on DPPH and hydroxyl radical in the same samples was determined on an ESR spectrometer. Fermentation broth and kombucha beverage from lemon balm had higher antioxidant activity against both radicals than the samples from black tea. Kombucha beverage from lemon balm with optimum acidity had higher antioxidant activity than unfermented lemon balm tea. The main active components of antioxidant activity were probably rosmarinic acid and quercetin. Antiproliferative activity of lemon balm tea and kombucha was measured by sulforhodamine B colorimetric assay on HeLa (cervix epitheloid carcinoma), HT-29 (colon adenocarcinoma), and MCF-7 (breast adenocarcinoma) cell lines. By applying concentrations higher than 100 &mu;g/ml, tested samples did not stimulate proliferation of cell lines. The possibility of simultaneous lactic acid and kombucha fermentation was tested as well. When starter cultures and Lactobacillus spp. isolates were applied, the content of Land D- lactic acid increased during fermentation, although lactic acid bacteria (except <em>L. hilgardii</em> isolated from sour dough) showed low resistance to the conditions during fermentation and beverages storage. Acetic acid bacteria isolated from local tea fungus were identified by PCR method. Two isolates might be <em>Gluconobacter oxydans</em> and one - <em>Gluconacetobacter hansenii.</em></p>