The use of Lactic Acid Bacteria (LAB), such as Lactococcus lactis (LL), as DNA delivery vehicles represents an interesting strategy as they are regarded as safe. Wild type (wt) LL or recombinant invasive LL, were able to trigger DNA expression by epithelial cells both in vitro and in vivo. However, important information about how LL can transfer DNA plasmids is still missing. Therefore, we decided to construct a new recombinant invasive LL strain expressing mutated Internalin A (mInlA) from the pathogen Listeria monocytogenes to understand the manner by which the DNA is transferred to mammalian cells. mInlA expression was detected by FACS analysis and LL-mInlA strain showed to be more invasive than the wt strain after co-incubation assays with non-confluent or polarized intestinal epithelial cells (IECs). Confocal microscopy confirmed the invasive status of LL-mInlA which demonstrated to deliver more efficiently the eukaryotic expression vector coding the allergen β-lactoglobulin, pValac:BLG, in vitro to IECs and to dendritic cells (DCs). LL-mInlA was also capable to transfer pValac:BLG to DCs across a monolayer of differentiated IECs. In vivo, invasive lactococci tended to increase the number of mice expressing BLG. Moreover, noninvasive or invasive LL-mInlA stimulated the secretion of the pro-inflammatory cytokine IL-12 in DCs and, in vivo, after oral or intranasal immunization trials, non-invasive LL polarized the immune response more in the type 1 direction while invasive LL generated a Th2-type response in immunized animals. All these data gives new insights on the mechanism of lactococci uptake for delivery of therapeutics.

L’ocratossina e le ammine biogene sono due metaboliti biologici che possono essere ritrovati nei vini. Il primo di questi contaminanti è stato studiato recentemente per la sua elevata tossicità sebbene non sia rintracciabile frequentemente nei vini. Le ammine biogene sono presenti in ogni tipo di vino in differenti concentrazioni, oltretutto, alcune di loro, ad alte concentrazioni, possono causare reazioni allergiche. Gli obiettivi di questo PhD riguardano tre differenti aspetti. Indagare metodi biologici di per la decontaminazione di ocratossina A durante la vinificazione; in particolare lo studio si è focalizzato sull’uso di un ceppo Lactobacillus plantarum utilizzato come starter malolattico. Valutare la presenza di Lactobacillus spp., isolati da mosto e vino, in grado di produrre ammine biogene, usando tecniche molecolari come la reazione a catena della polimerasi (PCR) per rilevare i geni codificanti gli enzimi responsabili della sintesi di questi composti. Testare la capacità di un L. plantarum di effettuare la malolattica effettuando l’inoculo in differenti fasi della vinificazione e valutare il trend delle ammine biogene già presenti nel mosto. / Two of the major biological metabolites present in wine are the ochratoxin and the biogenic amines. The first of these contaminants was studied in recent decades because of its toxicity in humans, although its presence is not frequent in wines. The biogenic amines are present in every types of wine in different concentration, and some of them, in high concentrations, can cause allergenic reactions in humans. The objectives of this PhD regard three different aspects. Investigate the biological methods to reduce ochratoxin A in wine during winemaking; in particular the study is focused to use a Lactobacillus plantarum strain as malolactic starter. Investigate the presence of Lactobacillus spp., isolated from must and wine, able to produce the amines, using molecular techniques as polymerase chain reaction (PCR) to detect the genes that encode for the enzymes responsible of the synthesis of these compounds. Test the ability of a L. plantarum to perform MLF in relationship with inoculation time and assess the trend of biogenic amines already present in must.

Šio darbo tikslas: nustatyti kietafaze fermentacija fermentuotų topinambų ir lubinų raugų įtaką mėsos pusgaminių kokybei ir saugai Darbo uždaviniai: Įvertinti kietafaze fermentacija (toliau – KF) fermentuotų skirtingomis pienarūgštėmis bakterijomis topinambų įtaką: kiaulienos pusgaminių fizikiniams – cheminiams rodikliams; jautienos pusgaminių fizikiniams – cheminiams rodikliams; lakiųjų junginių pokyčiams jautienos pusgaminiuose. Įvertinti KF fermentuotų skirtingomis pienarūgštėmis bakterijomis lubinų įtaką: kiaulienos pusgaminių fizikiniams – cheminiams rodikliams; jautienos pusgaminių fizikiniams – cheminiams rodikliams; lakiųjų junginių pokyčiams kiaulienos ir jautienos pusgaminiuose. Įvertinti KF fermentuotų skirtingomis pienarūgštėmis bakterijomis topinambų ir lubinų įtaką kiaulienos pusgaminių bendram bakteriniam užterštumui. Eksperimentui atlikti buvo gaminami kiaulienos ir jautienos pusgaminiai su 5 proc. raugu (fermentuotais lubinais ir topinambais, fermentacijai panaudojant tris pienarūgštes bakterijas: P. acidilactici KTU -05-7, P. pentosaceus KTU -05-8, L. sakei KTU -05-6). Naudota metodika: LST ISO 1442:2000 „Mėsa ir mėsos produktai. Drėgmės kiekio nustatymas (pamatinis metodas).; Grau ir Hammo (1956) metodas (vandens rišlumo nustatymas); Soksleto (1879) metodas (riebalų kiekio nustatymas); LST ISO 936:2000 „Mėsa ir mėsos produktai. Bendrojo pelenų kiekio nustatymas; lakiųjųi junginių analizė atlikta dujų chromatografu metodu. Išvados: Fermentuoti lubinų... [toliau žr. visą tekstą] / The aim of this paper is to identify the solid state fermentation of Jerusalem artichoke and lupine cultures affect to the quality and safety of meat ready-to-cook. Job tasks: Evaluate the solid state fermentation (thereinafter – SSF) fermente by lactic acid bacteria (thereinafter – LAB) in different Jerusalem artichoke influence: pork ready-to-cook physical- chemical parameters; beef ready-to-cook physical-chemical parameters; changes of volatile compounds in beef ready-to-cook. Evaluate the SSF of fermented in different LAB lupine influence: pork ready-to-cook physical- chemical parameters; beef ready-to-cook physical-chemical parameters; changes of volatile compounds in pork and beef ready-to-cook. Evaluate SFF ferment in different LAB Jerusalem artichoke and lupine influence to pork ready-to-cook impurity. In this experiment pork and beef ready-to-cook have been used with 5 percent product (fermented lupine and Jerusalem artichoke, for fementation were used 3 LAB: P. acidilactici KTU -05-7, P. pentosaceus KTU -05-8, L. sakei KTU -05-6). Methods: LST ISO 1442:2000 Meat and meat products – Determination of moisture content (References method).;Grau and Hamm (1956); Soxlet (1879); LST ISO 936:2000 Meat and meat products – Determination of total ash.; Detection volatile compounds using gas chromatography method. Conclusion: Ferment lupine and Jerusalem artichoke products have reduced pH in pork and beef ready-to-cook. Beef ready-to-cook fermentated with Jerusalem artichoke... [to full text]

Tikslo įgyvendinimui buvo atlikta baltagūžių ir mėlynųjų kopūstų bei saldžiųjų raudonųjų ir geltonųjų paprikų fermentacija skirtingomis pieno rūgšties bakterijomis (PRB) (L. sakei, P. pentosaceus KTU-08, P. pentosaceus KTU-09 ir P. acidilactici). Tiriant fermentacijos proceso efektyvumą buvo įvertinta pH dinamika, gautuose produktuose nustatytas PRB kolonijas sudarančių vienetų skaičius grame produkto (KSV/g), L (+) ir D-(-) pieno rūgšties izomerų kiekis, atlikta juslinė analizė, priimtiniausiems produktams įvertintos spalvų koordinatės ir tekstūros savybės. Pagal gautus tyrimų rezultatus galima teigti, kad eksperimente naudotos PRB yra tinkamos baltagūžių ir mėlynųjų kopūstų bei saldžiųjų raudonųjų ir geltonųjų paprikų fermentacijai. PRB taikymas daržovių fermentavimui yra efektyvus, nes į smulkintas daržoves įterpus PRB, jos kurį laiką dominuoja. Naudojant fermentacijai PRB, gaunamas pakankamai žemas produktų pH, o tai suteikia fermentuotoms daržovėms priimtinas vartotojams juslines savybes. Daržovių fermentacijai taikant PRB, gaunami didesnio šviesumo, raudonumo, geltonumo, grynumo bei spalvos tono produktai. Siekiant užtikrinti fermentuotų produktų saugą D(-) pieno rūgšties izomerų aspektu daržovių fermentacijai reikėtų rinktis L. sakei. / Fermentations with different lactic acid bacteria (LAB) (L. sakei, P. pentosaceus KTU-08, P. pentosaceus KTU-09 and P. acidilactici) of white cabbage, red cabbage, sweet red and yellow paprika were performed in order to achieve the goal. While studying the effectiveness of the fermentation process, pH dynamics was evaluated, colony forming units of LAB in fermented products, concentration of lactic acid isomers L (+) and D(-), sensory analysis, colour coordinates and texture parameters were evaluated. We conclude that in the experiment used LAB are suitable for the fermentation of a white cabbage, red cabbage, sweet red and yellow paprika. The application of LAB is effective, because LAB were dominate for some time in the fermented vegetables. While using LAB for the vegetables fermentation, pH was found quite low, and improve sensory properties of the fermented vegetables products. LAB gives higher brightness, clarity of red and yellow, as well as colour tone of the fermented products. In order to ensure the safety of fermented products, in the context of D (-) lactic acid, L. sakei would be more suitable for the vegetable fermentation.

Darbo tikslas: ištirti apsauginių kultūrų panaudojimo mėsos pusgaminių gamyboje galimybes, gerinant saugą ir kokybę. Darbo uždaviniai: įsisavinti apsauginių kultūrų panaudojimo mėsos pusgaminių gamyboje būdus, pusgaminių kokybinių ir saugos rodiklių nustatymo metodus; ištirti apsauginių bakterijų kultūrų: Lactobacillus spp. Staphylococcus xylosus, poveikį mėsos pusgaminių mikrobiologiniams ir fizikiniams cheminiams rodikliams, įvertinti jų saugą ir kokybę; ištirti termiškai apdorotų mėsos pusgaminių su apsauginėmis bakterinėmis kultūromis Lactobacillus spp., Staphylococcus xylosus juslines savybes; palyginti įprastų ir gautų mėsos pusgaminių su apsauginėmis kultūromis kokybę ir saugą, juslines savybes, vartojimo terminą; apdoroti statistiškai gautus duomenis, apibendrinti rezultatus ir pateikti išvadas. Rezultatai: priedas su apsauginėmis kultūromis Lactobacillus spp., Staphylococcus xylosus visiškai išnaikino E. Coli jautienos farše, supakuotame į vakuuminę pakuotę (p ≤ 0,01), modifikuotų dujų atmosferą (p ≤ 0,01), laikomo aerobinėmis sąlygomis (p ≤ 0,001) lyginant su kontrole. Apsauginių kultūros ženkliai sumažino bendrą aerobinių mikroorganizmų skaičių lyginant su kontrole (p ≤ 0,001). Apsauginių kultūrų įterpimas sumažino biogeninių aminų tiramino, spermidino ir spermino kiekius iki mažesnių už aptikimo ribą kiekių (< 5 mg/kg), tačiau mėginiuose su apsauginių kultūrų priedu, laikomuose aerobinėmis sąlygomis, nustatytas padidėjęs fenilalanino kiekis, lyginant su kontrole... [toliau žr. visą tekstą] / The aim of the work was to investigate the possibilities of using protective cultures in the manufacture of semi-finished meat in improving safety and quality. The tasks of the work were to master ways of using protective cultures in production of semi-finished meat; methods for determining quality and safety indicators of semi – finished meat; to investigate the impact of protective bacterial cultures: Lactobacillus spp., Staphylococcus xylosus to microbiological and physico-chemical parameters of semi – finished meat in order to ensure their safety and quality; to investigate organoleptic properties of the heat-treated semi – finished meat with a protective bacterial cultures Lactobacillus spp., Staphylococcus xylosus; to compare quality and safety, organoleptic characteristics, expiry date of conventional and meat preparations with protective cultures; to process statistically obtained data, summarize the results and present findings. Results: • Additive with protective cultures Lactobacillus spp., Staphylococcus xylosus completely destroyed E. coli in vacuum packed minced beef (p ≤ 0,01), in modified gas atmosphere (p ≤ 0,01), stored under aerobic conditions (p ≤ 0,001) compare with the controls. • Protective cultures significantly reduced the total number of aerobic microorganisms compare with controls (p ≤ 0,001). • Insertion of protective cultures reduced biogenic amines tyramine, spermidine and spermine below the detection limit (<5 mg / kg), however, samples... [to full text]

Thesis (MSc Food Sc )--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Kepi is a refreshing, fermented dairy beverage that differs from other fermented milk products in that it is produced with a mixed microbial community which is confined to discrete grains. These grains can be recovered as a solid matrix at the end of the fermentation and then be reutilised as a starter to ferment the next batch of milk. The grain microbial community consists of a symbiotic association of yeasts and lactic acid bacteria, but the overall composition of the grains has not been completely elucidated. The microbes in the grains are embedded in a protein-polysaccharide Kefiran matrix, which appears essential for grain formation. The mechanism of grain formation is still not fully understood and it thus remains undecided which organism is really responsible for the production of this proteinpolysaccharide matrix. The aim of this study was to isolate, characterise and identify the microbes present in Kefiran from mass cultured South African grains and then to evaluate grain formation with these purified cultures isolated from Kefiran strings using a mass cultivation process. Sixteen strains of lactic acid bacteria and one yeast strain were isolated from Kefiran strings produced during the mass cultivation of South African Kepi grains. API technology, numerical clustering and DNA sequence comparisons were used to identify the purified isolates. The isolates were grouped into seven clusters by numerical clustering and clustering distance from selected reference and marker strains. The heterofermentative lactobacilli were identified as Lactobacillus parakefiri and Lb. kefiri and the homofermentative strains as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus and Lb. bavaricus. One isolate was found to be a member of the genus Lactobacillus, but was not positively identified to species level. Cultures isolated from Kefiran were evaluated for ability to grain formation by adding 1 x 109 cfu.ml:' bacteria and 1 x 108 cfu.ml' yeast to double pasteurised, full cream milk during the mass cultivation process. It was found that the control and all the cultures in double pasteurised milk showed grain accumulation indicating that other microbes were present in pasteurised and double pasteurised milk which had an influence on the grain forming ability. The cultures isolated from pasteurised and double pasteurised milk included members of the species Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliermondii, Chryseobacterium meningosepticum, Pseudomonas putida and four isolates of the Bacillus cereus group. It was found that these rod-shaped "milk isolates" resulted in grain accumulation when inoculated into UHT milk and it was concluded that the "milk isolates" did contribute to grain formation. These isolates were then combined with the Kefiran cultures and this resulted in grains very similar to the traditional Kepi grains. These grains were made from Lb. gallinarum in double pasteurised milk as well with a combination of Lb. gallinarum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica and Pseudomonas putida in URT milk. The grains were firm, elastic and did not dissolve in water but kept their structure and were retained when sieved. An acceptable Kepi beverage was produced from these grains. From these typically traditional grain characteristics it was concluded that, even though the microbial compositions were probably not the same, the general appearance was similar to traditional grains and that it is thus possible to produce grains from pure single strain Kefiran cultures and "milk isolates". Furthermore, it was possible to produce a Kepilike beverage from these grains, which included similar characteristics as the traditional Kepi beverage. / AFRIKAANSE OPSOMMING: Kepi is "n verfrissende, gefermenteerde suiweldrankie wat van ander gefermenteerde produkte verskil in die opsig dat dit vervaardig word deur Kepi korrels in melk te inkubeer. Die Kepi korrels kan aan die einde van die fermentasie herwin word en weer gebruik word om die volgende lot melk te fermenteer. Die korrels bestaan uit "n simbiotiese samestelling van giste en melksuurbakterieë, maar die presiese samestelling van die korrels is steeds onbekend. Die mikro-organismes is vasgevang in "n proteïen-polisakkaried Kefiran matriks en die Kefiran word as essensieel beskou vir korrelvorming. Die meganisme van korrelvorming bly steeds onbekend en daar is nog nie tot "n gevolgtrekking gekom oor watter organisme die Kefiran produseerder is nie. Die doel van die studie was om die mikro-organismes in Kefiran te isoleer en te identifiseer deur Suid-Afrikaanse Kepi korrels te massa kweek. Hierdie mikroorganismes was dan verder geëvalueer ten opsigte van korrel vorming. Sestien melksuurbakterieë isolate en een gis isolaat is geïsoleer vanuit die Kefiran. API tegnologie, numeriese groepering en DNA volgorde vergelykings was gebruik om die isolate te identifiseer. Die isolate is in sewe groepe verdeel volgens numeriese groepering. Die afstand van verwysings en merker organismes is ook in ag geneem. Die heterofermentatiewe organismes is geïdentifiseer as Lactobacillus parakefiri en Lb. kefiri en die heterofermentatiewe organismes as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus en Lb. bavaricus. Een isolaat kon nie geïdentifiseer word tot op spesie vlak nie, maar is verwant aan die genus Lactobacillus. Hierdie geïsoleerde Kefiran kulture is geëvalueer ten op sigte van korrelvorming, deur 1 x 109 kve.ml' van die bakterieë en 1 x 108 kve.ml' van die gis by dubbel gepasteuriseerde volroom melk te voeg tydens die massakwekings proses. Die kontrole wat geen bygevoegde kulture bevat nie, sowel as die wat wel bygevoegde kulture bevat, het korrel vorming getoon. Laasgenoemde toon dat daar organismes teenwoordig is in gepasteuriseerde en dubbel gepasteuriseerde melk wat "n rol kan speel tydens korrelvorming. Die kulture wat geïsoleer is vanuit gepasteuriseerde en dubbel gepasteuriseerde melk, sluit in: Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliennondii, Chryseobacterium menigosepticum, Pseudomonas putida en vier isolate van die Bacillus cereus groep. Hierdie organismes wat uit melk geïsoleer is, het korrelvorming getoon in UHT melk en die gevolgtrekking kan gemaak word dat die "melk organismes" wel "n rol speel tydens korrel vorming. Hierdie "melk isolate" in kombinasie met die Kefiran kulture het korrels tot gevolg gehad wat baie dieselfde was as tradisionele Kepi korrels. Laasgenoemde korrels is gemaak deur Lb. gallina rum in dubbel gepasteuriseerde melk, sowel as deur "n kombinasie van Lb. gallina rum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica en Pseudomonas putida in UHT melk. Die korrels was stewig, elasties, het nie opgelos in water nie en het hulle struktuur behou wanneer gesif. Wanneer hierdie tipiese tradisionele korrels se eienskappe in ag geneem word, kan die gevolgtrekking gemaak word dat alhoewel die mikrobiese samestelling van die korrels nie dieselfde is as die tradisionele korrel nie, is die algemene voorkoms en eienskappe dieselfde en dat dit wel moontlik is om korrels te produseer deur isolate geïsoleer vanuit Kefiran en melk. Verder was dit moontlik om "n drankie te vervaardig met die korrels wat baie dieselfde is as tradisionele Kepi.

A microbiota vaginal saudável é constituída, majoritariamente, por espécies de lactobacilos que representam uma barreira natural contra microrganismos causadores de doenças como a candidíase vulvovaginal (CVV), vaginose bacteriana (VB) e infecções do trato urinário (ITU), que juntas acometem cerca de um bilhão de mulheres no mundo anualmente. Um melhor entendimento da ecologia microbiana vaginal pode ser útil na otimização de tratamentos existentes para as infecções urogenitais, os quais podem destruir parcialmente a microbiota autóctone, predispor a novas infecções, contribuir para a seleção de microrganismos resistentes e causar efeitos colaterais indesejáveis. A utilização de microrganismos certificadamente probióticos, Lactobacillus rhamnosus GR-1 e Lactobacillus reuteri RC-14, representa uma alternativa terapêutica promissora na abordagem de VB e CVV, uma vez que são capazes de colonizar o trato vaginal, apresentam atividade inibitória frente a diversos patógenos do trato urogenital, exibem risco mínimo para a seleção de microrganismos resistentes e podem auxiliar na restauração da microbiota vaginal. Os objetivos deste trabalho foram: (i) avaliar a prevalência de espécies de lactobacilos na microbiota vaginal de mulheres saudáveis e diagnosticadas com infecções vaginais (CVV e VB) da cidade de Ribeirão Preto (São Paulo, Brasil), (ii) avaliar a capacidade de isolados de lactobacilos produzirem peróxido de hidrogênio (H2O2) e (iii) determinar a eficácia da utilização de L. rhamnosus GR-1 e L. reuteri RC-14 no tratamento de CVV e VB, quando co-administrados com medicamentos antimicrobianos tradicionais. Participaram deste estudo, 196 pacientes voluntárias, atendidas por médicos ginecologistas de centros de saúde ligados à Universidade de São Paulo, campus de Ribeirão Preto (64 saudáveis, 68 diagnosticadas com CVV e 64 diagnosticadas com VB) e duas amostras vaginais de cada paciente foram coletadas com o auxílio de zaragatoas esterilizadas. Uma zaragatoa foi utilizada para semeadura em ágar MRS (de Man, Rogosa & Sharpe), os isolados de bactérias láticas obtidos foram analisados através da técnica de PCR-ARDRA (Reação em cadeia da polimerase Análise de restrição do DNA ribossomal amplificado) e a habilidade de Lactobacillus spp. produzir H2O2 foi determinada semi-quantitativamente. A outra zaragatoa foi utilizada para a análise das espécies de lactobacilos da microbiota vaginal pela técnica independente de cultivo PCR-DGGE (Reação em cadeia da polimerase Eletroforese em gel de gradiente de desnaturação). As leveduras do gênero Candida foram obtidas através da semeadura das amostras vaginais provenientes de pacientes saudáveis e com CVV no meio Chromagar® Candida e identificadas através de provas bioquímicas de referência. As pacientes diagnosticadas com CVV participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de fluconazol (150mg) e suplementação diária durante 28 dias com (i) duas cápsulas contendo os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 (Urex-Cap-5®) ou (ii) duas cápsulas de placebo. As pacientes com VB também participaram de um estudo randomizado, duplo-cego, placebo-controlado e foram tratadas com dose única de tinidazol (2g) e suplementação diária com cápsulas do probiótico (Urex-Cap-5®) ou placebo, conforme descrito acima. Todas as pacientes foram reavaliadas ao final do tratamento, no 28º dia. Foram realizados experimentos para averiguar o possível efeito de L. rhamnosus GR-1 e L. reuteri RC-14 na modulação in vitro da infecção por Candida albicans em culturas de células epiteliais vaginais humanas (VK2/E6E7) Os resultados mostraram que, pela técnica de PCR-ADRA, L. crispatus foi a espécie mais prevalente nos grupos de pacientes saudáveis (37,0%) e com CVV (35,9%), enquanto que L. gasseri foi predominante no grupo de pacientes com VB (34,6%). De acordo com o método de PCR-DGGE, L. iners foi o microrganismo mais prevalente nos três grupos de pacientes avaliados: saudáveis, com CVV e VB (48,7%, 44,7% e 65,0%, respectivamente). A maioria dos isolados de Lactobacillus spp. obtidos nos grupos de pacientes saudáveis (98,6%) e diagnosticadas com CVV (97,4%) foram capazes de produzir H2O2 (1 a 100mg/L), em comparação a apenas 68,2%, determinado no grupo de pacientes com VB (p<0,05). L. crispatus e L. johnsonii produziram as maiores quantidades médias de H2O2 (30mg/L). A taxa de colonização por leveduras do gênero Candida foi de 26,6% no grupo de pacientes saudáveis (C. albicans correspondeu a 52,4% de todos os isolados), enquanto que no grupo de pacientes com CVV, 89,2% dos isolados leveduriformes foram identificados como C. albicans. Para as análises estatísticas dos dados obtidos com os testes clínicos, foram consideradas 55 pacientes diagnosticadas com CVV (pela presença de sinais e sintomas da infecção e cultura positiva para Candida sp.) e foi observado que a utilização de dose única de fluconazol e suplementação diária com o probiótico, resultou em taxa de cura mais elevada para a infecção (89,7%) em comparação com aquela verificada no grupo placebo (65,4%) (p<0,05). A utilização de tinidazol em associação com a ingestão diária de Urex-Cap-5® no tratamento de pacientes com VB também resultou em taxa de cura mais elevada para a condição (87,5%), em comparação àquela observada no grupo placebo (50,0%) (p<0,05). A atividade anti-Candida de L. rhamnosus GR-1 e L. reuteri RC-14 foi observada através do modelo in vitro para infecção vaginal. Em conclusão, quando as técnicas de PCR-ARDRA e PCR-DGGE foram comparadas entre si, foi observado que ambas apresentaram limitações, evidenciando a importância do emprego de diferentes metodologias para avaliação adequada das espécies de lactobacilos presentes na microbiota vaginal. As espécies de lactobacilos vaginais determinadas nas mulheres saudáveis do presente trabalho foram semelhantes àquelas verificadas em estudos prévios descritos na literatura com pacientes com dieta e localização geográfica notadamente distintas. Os dados do presente trabalho sugerem que a presença de lactobacilos produtores de H2O2, isoladamente, não confere proteção contra CVV, enquanto que a ausência desses microrganismos pode ser um fator contribuinte para VB. Além disso, foi demonstrado que a utilização de medicamentos antimicrobianos tradicionais e suplementação com os microrganismos probióticos L. rhamnosus GR-1 e L. reuteri RC-14 foi mais eficiente no tratamento de CVV e VB em comparação com medicamentos clássicos e placebo. Estes resultados podem contribuir para prolongar a vida útil de medicamentos cuja eficácia pode ser comprometida devido à seleção de microrganismos resistentes e também reduzir o tempo de tratamento para pacientes que necessitam de terapias clássicas por períodos prolongados. / The vaginal microbiota is mainly constituted by lactobacilli species, which represent a natural barrier against microorganisms that cause vulvovaginal candidiasis (VVC), bacterial vaginosis (BV), and urinary tract infections (UTI). Together, these conditions afflict each year an estimated one billion women worldwide. A better understanding of the vaginal microbial ecology may be useful to improve the current available treatments for urogenital infections, which can partially destroy the autochthonous microbiota, predispose to other infections, contribute for the selection of resistant microorganisms and cause undesirable collateral effects. The use of microorganisms with demonstrated probiotic properties, Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, represents a promising therapeutic alternative for BV and VVC, since they are able to colonize the vaginal tract, present inhibitory against several urogenital pathogens, pose minimal risk for the selection of resistant microorganisms and can help to restore the vaginal microbiota. The objectives of this work were: (i) to evaluate the prevalence of lactobacilli species in the vaginal microbiota of healthy women and those diagnosed with vaginal infections (VVC and BV) in the city of Ribeirão Preto (São Paulo, Brazil), (ii) to evaluate the ability of the lactobacilli isolates to produce hydrogen peroxide (H2O2) and (iii) to determine the efficacy of the use of L. rhamnosus GR-1 and L. reuteri RC-14 in the treatment of VVC and BV, in co-administration with traditional antimicrobials. 196 voluntary subjects were examined by the gynecologists team from health centers affiliated with Universidade de São Paulo, campus at Ribeirão Preto (64 healthy, 68 diagnosed with VVC and 64 diagnosed with BV) and two vaginal samples from each patient were collected by the use of two sterile swabs. One swab was cultured in MRS (de Man, Rogosa & Sharpe) agar, the isolates of lactic acid bacteria obtained were analyzed by PCR-ARDRA (Polymerase chain reaction - Amplified ribosomal DNA restriction analysis) and the ability of Lactobacillus spp. to produce hydrogen peroxide was determined semi-quantitatively. The other swab was used for the analysis of lactobacilli species from the vaginal microbiota using the culture-independent PCR-DGGE (Polymerase chain reaction Denaturating gradient gel electrophoresis). The yeasts belonging to Candida genus were obtained also by culturing the vaginal material from healthy and VVC patients in Chromagar® Candida and were identified by standard biochemical tests. VVC patients were enrolled in a randomized, double-blind, placebo-controlled trial and treated with a single dose fluconazole (150mg) and daily supplementation for 28 days with (i) two capsules containing the probiotic microorganisms L. rhamnosus GR-1 and L. reuteri RC-14 (Urex-Cap-5®) or (ii) two placebo capsules. BV patients were also enrolled in a randomized, double-blind, placebo controlled trial and treated with a single dose of tinidazole (2g) and supplementation with probiotic capsules (Urex-Cap-5®) or placebo, as described above. All patients were re-evaluated at the end of treatment, on the 28th day. Experiments were also conducted to assess the possible effect of L. rhamnosus GR-1 and L. reuteri RC-14 in the in vitro modulation of vaginal infection by Candida albicans on cultures of human vaginal epithelial cells (VK2/E6E7). The results revealed that according to PCR-ADRA, L.crispatus was the most prevalent species in the groups of healthy women (37.0%) and those with VVC (35.9%), while L. gasseri was dominant in BV patients (34.6%). By PCR-DGGE method, L. iners was the most prevalent Lactobacillus species in all the three groups evaluated: healthy, VVC and BV (48.7%, 44.7% and 65.0%, respectively). The majority of the isolates of Lactobacillus spp. from healthy women (98.6%) and those with VVC (97.4%) were able to produce H2O2 (1 to 100mg/L) in comparison with only 68.2% assessed for the BV group (p<0.05). L. crispatus and L. johnsonii produced the highest average levels of H2O2 (30mg/L). Colonization rate by yeasts belonging to Candida genus was 26.6% in the group of healthy patients (C. albicans represented 52.4% of all isolates), whereas in the VVC group, 89.2% of yeast isolates were identified as C. albicans. For the performance of statistical analysis of the results obtained with the clinical trials, 55 patients diagnosed with VVC (by the presence of symptoms and signals of the infection and positive culture for Candida sp.) were taken into consideration and it was observed that the use of a single dose of fluconazole and daily supplementation with probiotics, yielded a higher cure rate (89.7%), in comparison with the placebo group (65.4%) (p<0.05). The use of tinidazole plus probiotic also resulted in higher cure rate of the infection (87.5%), compared to placebo group (50.0%) (p<0.05). An anti-Candida activity of L. rhamnosus GR-1 and L. reuteri RC-14 was observed in the in vitro model of vaginal infection. In conclusion, when PCR-ARDRA and PCR-DGGE were compared, it was verified that both presented limitations, which evidences the need of using different techniques for a better knowledge of lactobacilli species present in the vaginal microbiota. The lactobacilli species found in healthy women in this work were similar to those reported in previous studies described in the literature for patients with distinctly different diet and geographic localization. The data of the present work indicate that solely the presence of H2O2-producing isolates does not render protection against VVC, whereas the absence of those microorganisms may be a contributing factor for BV. Moreover, it was demonstrated that the use of classical medicines supplemented with the probiotics L. rhamnosus GR-1 and L. reuteri RC-14 was more efficient to treat VVC and BV in comparison with classical medicines plus placebo. These results may contribute to extend the longevity of drugs whose efficacy is compromised due to the selection of resistant microorganisms and also to shorten the length of treatment courses for patients that require long regimens with standard therapy.

Made available in DSpace on 2015-04-17T14:49:17Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 884266 bytes, checksum: b2fc6505d10abdbaaf135855e8e6fd2c (MD5) Previous issue date: 2009-09-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Researches demonstrate that the consumption of fermented milk is beneficial to the health due to the presence of lactic acid bacteria and of the products of the metabolism produced by them during the fermentation of the milk. The objective of this work was to evaluate the action of the lactic bacterias of two commercial marks of fermented milks, one cultivated with Lactobacillus paracasei (L1) and other with Lactobacillus casei (L2), on the weight gain and hematologic and histopathologic parameters of rats Wistar making the indomethacin use. The choice of the drug based on the hypothesis of the existence of the protection of the digestive system and of the histology of the organs of the animals against the possible aggression of the nonsteroidal anti-inflammatory for the concomitant administration of the fermented milks. Three lots of each fermented milk were used, taking place the count of the viability of the lactic bacterias, gram coloration, catalase and morphologic identification. Simultaneously, 60 rats Wistar males with 90 days, consuming commercial ration and water ad libitum, were divided in 6 groups of 10, being the group LP added of L1; LP + D of L1 + drug; C (control) of water; D, of drug; LC, of L2; and LC + D, of L2 + drug. The animals received the fermented milks (5mL/Kg/day) and/or the drug indomethacin (2mg/Kg/day) for gavage for 40 days, enrolling the weekly weights. After the sacrifice, blood samples were collected for the accomplishment of hemograms and it was verified the weight of the kidneys, spleens and livers, taking place the histopathology of these organs and of the stomachs and intestines. The obtained results were treated with ANOVA, Tukey, Friedman, Duncan, Kruskall-wallis and Wilcoxon (p<0,05), being used the statistical package R. The strains of L1 and L2 resulted in catalase negative, gram positive and morphology of bacilli, presenting final counts of bacteria with superior values to 106 UFC. The fermented milks added to the diets of LP and LC and the addition of the drug to D didn't influence in the weekly weight gain of the animals, but LP+D and LC+D statistically differed of C and D, suggesting there to be interaction among drug-food. In the difference among the initial and final weight of the animals, the groups that received fermented milk resembled each other to the control, except the group LP whose weight gain was inferior; D also presented deficit of weight gain in relation to the group C. The diets didn't influence in the weights of the kidneys of the experimental groups, LP just presented significant difference in terms of spleen weight in relation to the other groups and the weights of the livers of the groups LP, D and LC+D differed in relation to the control group, where D resulted in superior weight of C and LP and LC+D obtained inferior weights to the control group. In the blood parameters, the groups didn't differ to each other in the red and white series, nor in the differential leucocyte count, if not verifying immunostimulatory effects; already in the counts of the platelets, some groups presented statistical difference, however the obtained results were inside of the allowed. The histopathological analysis had not evidenced histology alterations in the stomachs, livers, kidneys and spleens, meeting discreet infiltrated of lymphoid cells in the own sheet of the intestine of the experimental groups. / Pesquisas demonstram que o consumo de leite fermentado é benéfico à saúde devido à presença de bactérias láticas e dos metabólitos produzidos por elas durante a fermentação do leite. O objetivo deste trabalho foi avaliar a ação das bactérias láticas de duas marcas comerciais de leites fermentados, uma cultivada com Lactobacillus paracasei (L1) e outra com Lactobacillus casei (L2), sobre o ganho de peso e parâmetros hematológicos e histopatológicos de ratos Wistar fazendo o uso de indometacina. A escolha da droga baseou-se na hipótese da existência da proteção do trato digestivo e da histologia dos órgãos dos animais contra a possível agressão do antiinflamatório pela administração concomitante dos leites fermentados. Foram utilizados três lotes de cada leite fermentado, realizando-se a contagem da viabilidade das bactérias láticas, coloração de gram, catalase e identificação morfológica. Simultaneamente, 60 ratos Wistar machos com 90 dias, consumindo ração comercial e água ad libitum, foram divididos em 6 grupos de 10, sendo o grupo LP adicionado de L1; LP + D de L1 + droga; C (controle) de água; D, de droga; LC, de L2; e LC + D, de L2 + droga. Os animais receberam os leites fermentados (5mL/Kg/dia) e/ou a droga indometacina (2mg/Kg/dia) por gavagem durante 40 dias, registrandose os pesos semanais. Após o sacrifício, amostras sanguíneas foram coletadas para a realização de hemogramas e verificou-se o peso dos rins, baços e fígados, realizando-se a histopatologia destes órgãos e dos estômagos e intestinos. Os dados obtidos foram tratados com ANOVA, Tukey, Friedman, Duncan, Kruskall-wallis e Wilcoxon (p<0,05), utilizando-se o pacote estatístico R. As cepas de L1 e L2 resultaram em catalase negativas, gram positivas e morfologia de bacilos, apresentando contagens finais de bactérias com valores superiores a 106 UFC/mL. Os leites fermentados adicionados às dietas de LP e LC e a adição da droga ao grupo D não influenciaram no ganho de peso semanal dos animais, mas LP+D e LC+D diferiram estatisticamente de C e D, sugerindo haver interação entre droga-alimento. Na diferença entre o peso inicial e final dos animais, os grupos que receberam leite fermentado assemelharam-se ao controle, exceto o grupo LP cujo ganho de peso foi inferior; D também apresentou déficit de ganho de peso em relação ao grupo C. As dietas não influenciaram nos pesos dos rins dos grupos experimentais, apenas LP apresentou diferença significativa em termos de peso de baço em relação aos demais grupos e os pesos dos fígados dos grupos LP, D e LC+D diferiram em relação ao grupo controle, onde D resultou em peso superior a C e LP e LC+D obtiveram pesos inferiores ao grupo controle. Nos parâmetros sanguíneos, os grupos não diferiram entre si nas séries vermelha e branca, nem na contagem diferencial dos leucócitos, não se constatando efeito imunoestimulador; já nas contagens das plaquetas, alguns grupos apresentaram diferença estatística, porém os resultados obtidos encontraramse dentro da faixa permitida. As análises histopatológicas não evidenciaram alterações na histologia dos estômagos, fígados, rins e baços, apresentando discreto infiltrado de células linfóides na lâmina própria do intestino dos grupos experimentais.

No description available.

Bakgrund: Med industrialiseringen har de traditionella yoghurtkulturerna med en mångfald av samverkande mjölksyrabakterier fått ge plats åt de mer standardiserade. En gårdskultur framställs genom att obehandlad mjölk spontanfermenteras av gårdens egen bakterieflora och får därigenom en unik karaktär. Bakteriekulturen kan sedan ympas vidare för tillverkning av yoghurt.Syfte: Syftet med studien är framställning och vidareympning samt sensorisk och mikrobiologisk karaktärisering av termofil gårdskultur från obehandlad mjölk. Vidareympningen avser framställning av yoghurt fermenterad av gårdens egen bakterieflora.Metod: Gårdskulturen framställdes och ympades till yoghurt. Yoghurten undersöktes genom mikrobiologisk karaktärisering, antibiotikaresistens och sensorisk profilering samt jämfördes med industriell kultur och en traditionell heirloomkultur.Resultat: Resultatet visade att gårdskulturen skiljde sig både mikrobiologisk och sensoriskt. Gårdskulturen innehöll stammar av enterokocker vilka inte visade på resistens mot analyserade antibiotika.Slutsats: Det är möjligt att framställa en gårdskultur av godtagbar mikrobiologisk och sensorisk kvalitet. Gårdskulturen ger en differentierad mikrobiologisk och sensorisk karaktär i jämförelse med en industriell kultur och en traditionell heirloomkultur Metoden kan vara riskfylld och kulturen bör analyseras med avseende på patogen tillväxt. En unik gårdsyoghurt kan vara en metod för gårdsmejerister att i sitt varumärke bygga på terroir och platsens unicitet. / Background: With industrialization, the traditional yoghurt cultures with a multitude of lactic acid bacteria had to make way for the more standardized. An artisanal farm culture is produced by raw milk spontaneously fermented by the farm's own bacterial flora and thus develops a unique character. The bacterial culture can then be inoculated for the production of yoghurt.Purpose: The pupose of the study is to produce and inoculate as well as sensory and microbiological characterization of a thermophilic artisanal farm culture from raw milk. The inoculation relates to the production of yoghurt fermented by the farm's own bacterial flora.Method: The artisanal farm culture was produced and inoculated into yoghurt which was assessed by microbiological characterization, antibiotic resistance, sensory profiling and then compared with industrial culture and a traditional heirloom culture.Result: The result showed that the artisanal farm culture differed both microbiologically and with regard to sensory paramters. The farm culture contained strains of enterococci which did not show resistance to analyzed antibiotics.Conclusion: It is possible to produce an artisanal farm culture of good microbiological and sensory quality. The artisanal farm culture provides a differentiated microbiological and sensory character in comparison to an industrial culture and a traditional heirloom culture The method may be risky and the culture should be analyzed for pathogens. A unique farm yoghurt can be a method for artisan farm dairies to build their brand based on terroir.