Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Ciência dos Alimentos. / Made available in DSpace on 2013-07-15T23:32:21Z (GMT). No. of bitstreams: 0

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências dos Alimentos, Florianópolis, 2013. / Made available in DSpace on 2013-12-05T23:31:48Z (GMT). No. of bitstreams: 1 320589.pdf: 2081959 bytes, checksum: 7cf922f884890474ce3163ddea7491c2 (MD5) Previous issue date: 2013 / Este trabalho visou primeiramente o desenvolvimento e caracterização de microcápsulas contendo o micro-organismo probiótico Bifidobacterium BB-12, microencapsulado com substituição parcial (1:1) de leite desnatado reconstituído (LDR) por agentes prebióticos (inulina, inulina enriquecida com oligofrutose e oligofrutose) através da técnica de spray drying. A caracterização das microcápsulas compreendeu as seguintes análises: viabilidade, morfologia, tamanho de partícula, umidade, atividade de água, solubilidade, higroscopicidade, cor, propriedades térmicas, rendimento, sobrevivência em condições gastrintestinais simuladas e após tratamento térmico. Posteriormente, as microcápsulas que apresentaram o melhor desempenho nas análises anteriores foram aplicadas em creme de ricota, e o produto foi avaliado quanto às propriedades microbiológicas, físicas, químicas e sensoriais. A partir dos resultados obtidos, pôde-se observar uma elevada sobrevivência da bifidobactéria microencapsulada, tanto durante o processo de spray drying, como ao longo do armazenamento, sendo que as maiores contagens foram observadas nas microcápsulas produzidas com LDR e inulina enriquecida com oligofrutose. Além disso, de maneira geral, os resultados da caracterização física sugerem uma maior estabilidade para as microcápsulas produzidas com prebióticos. A utilização dos prebióticos inulina e inulina enriquecida com oligofrutose melhorou o rendimento da encapsulação, além de conferir maior proteção contra as condições gastrintestinais simuladas e os tratamentos térmicos e, por isso, as formulações contendo estes prebióticos foram escolhidas para a aplicação em creme de ricota. As contagens ao longo do armazenamento para as amostras de creme de ricota com bifidobactéria microencapsulada foram maiores do que para as amostras com células livres, sendo que as microcápsulas produzidas com LDR e inulina mostraram a maior viabilidade. Os valores de sólidos totais e firmeza foram maiores para as amostras com microcápsulas. Todas as amostras apresentaram um perfil similar para os valores de pH, acidez, sólidos totais e cor. De acordo com os julgadores, todas as amostras de creme de ricota apresentaram boa aceitabilidade sensorial. Desta forma, os resultados obtidos neste trabalho sugerem que a microencapsulação por spray drying pode ser usada como um efetivo método para manter a sobrevivência de Bifidobacterium BB-12, e que a combinação dos agentes encapsulantes LDR e inulina foi a mais indicada para a aplicação em creme de ricota <br>

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-06-25T16:48:22Z No. of bitstreams: 1 texto completo.pdf: 1893649 bytes, checksum: 331fc7abfe38b077d8aacb8db5668851 (MD5) / Made available in DSpace on 2018-06-25T16:48:22Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1893649 bytes, checksum: 331fc7abfe38b077d8aacb8db5668851 (MD5) Previous issue date: 2013-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os leites fermentados são ideais carreadores de micro-organismos probióticos devido a seu histórico de consumo e fabricação. A aplicação de bifidobactérias em produtos lácteos probióticos é geralmente associada a outras bactérias fermentadoras, com objetivos de garantir a qualidade tecnológica e sensorial do produto. A microbiota mista dos leites fermentados deve ser analisada diferencialmente com uso de meios seletivos para garantir a viabilidade e concentração de células. Metodologias alternativas rápidas para enumeração de micro-organismos, como o PetrifilmTM, são vantajosas para a indústria de alimentos, porém requerem prévia padronização. O objetivo desse trabalho foi adaptar meios seletivos para bifidobactérias com plaqueamento em PetrifilmTM AC. Cepas de bifidobactérias, lactobacilos e estreptococos foram pesquisadas em relação ao perfil de fermentação de carboidratos, redução e inibição por cloreto de 2,3,5- trifeniltetrazólio (TTC) e desenvolvimento em meios seletivos para bifidobactérias. Adicionalmente, o meio de cultura Rafinose-Propionato-MUP (RP-MUP) com plaqueamento convencional e em PetrifilmTM AC com diferentes tempos de incubação foi comparado com a metodologia da ISO29981 com meio TOS-MUP em leites fermentados produzidos. A rafinose foi o carboidrato que apresentou maior especificidade de fermentação por bifidobactérias, sendo considerada a fonte de carbono a ser utilizada no desenvolvimento do meio de cultura alternativo RP-MUP. Todas as cepas de Bifidobacterium spp. conseguiram reduzir adequadamente o TTC, e não foram inibidas nas concentrações 25, 50 e 100 mg/L. TOS-MUP e RP-MUP foram os meios de cultura que apresentaram melhor seletividade para bifidobactérias, quando comparadas aos meios MRS-ABC e MRS-NNLP. A associação do RP-MUP a placas PetrifilmTM AC permitiu a enumeração seletiva de bifidobactérias em leites fermentados, com equivalência de resultados com o RP-MUP e TOS-MUP utilizados pela metodologia convencional de plaqueamento (ANOVA, p < 0.05), e altos índices de correlação (r = 0.99, p < 0.05), inclusive em um tempo inferior de incubação (48 h). O protocolo alternativo proposto para enumeração de bifidobactérias em leites fermentados apresentou desempenho adequado, e representa uma vantagem para utilização pelas indústrias de alimentos no controle de qualidade de produtos probióticos com esses micro-organismos. / Fermented milks are ideal carriers of probiotic microorganisms due to their consumption and production history. The application of bifidobacteria in probiotic dairy products is usually associated with other fermenting bacteria, with the purpose of ensuring the technological and sensory qualities of the product. The mixed microbiota of fermented milks must be screened differentially with the usage of selective media to ensure the viability and concentration of the cells. Rapid alternative methodologies for the enumeration of microorganisms, like PetrifilmTM, are advantageous to the food industry, but they need previous standardization. The aim of this study was to adapt selective media for bifidobacteria with PetrifilmTM AC platting. Bifidobacteria, lactobacilli and streptococci strains were screened for carbohydrate fermentation profile, reduction and inhibition by 2,3,5-triphenyltetrazolium chloride (TTC) and growth in selective media for bifidobacteria. Additionally, Raffinose-Propionate-MUP (RP-MUP) with conventional platting and using PetrifilmTM AC with different incubation times was compared to ISO29981 methodology with TOS-MUP medium in fermented milks produced. Raffinose was the carbohydrate that presented the higher fermentation specificity by bifidobacteria, being considered for the development of alternative selective medium RP-MUP. All bifidobacteria strains were able to adequately reduce TTC, and they were not inhibited by concentration of 25, 50 and 100 mg/L. TOS-MUP and RP-MUP were the culture media with best selectivity for bifidobacteria, when compared to MRS-ABC and MRS-NNLP media. RP-MUP and PetrifilmTM AC plates association allowed selective enumeration of bifidobacteria in fermented milks, with equivalent results for RP-MUP and TOS-MUP used with conventional platting methodology (ANOVA; p < 0.05), and high correlation indexes (r = 0.99; p < 0,05), even with shorter incubation time (48 h). The suggested alternative protocol for the enumeration of bifidobacteria in fermented milks presented adequate performance, and represents an advantage for the usage by food industries in the quality control of probiotic products with these microorganisms.

La présente étude vise à étudier le potentiel de bifidobactéries à protéger les cellules contre l’infection par le Coxsackie B4 (CV-B4). Le criblage de bifidobactéries a identifié deux des cinq souches qui protégeaient les cellules HEp-2 lorsque les bifidobactéries sont pré-incubées avec les particules virales avant l'inoculation sur les cellules Hep-2. En revanche, aucun effet protecteur n'a été observé en incubant les cellules Hep-2 avec des bifidobactéries avant l'inoculation de CV-B4. Les lipoprotéines des parois cellulaires (LpAs) sécrétées par les souches sélectionnées sont testées pour leur activité antivirale. Les deux LpAs présentaient une activité antivirale quand ils sont incubés avec les particules virales avant d’être inoculées aux cellules HEp-2. Aucun effet protecteur n'a été induit par incubation des LpAs avec les cellules HEp-2 avant l'inoculation de CV-B4. La protéine recombinante présente une activité antivirale identique. Pour identifier les séquences peptidiques interagissant avec les particules virales, les protéines de LpAs sont alignées avec les séquences peptidiques du nord bord du canyon et avec l’empreinte de la région puff sur le Coxsackievirus et sur le récepteur de l’adénovirus (CAR). L'étude d'amarrage moléculaire in silico (Docking) utilisant le CV-B3 en tant que modèle a montré une faible énergie de liaison indiquant un système stable pour les peptides sélectionnés et par conséquent une interaction probable avec le CV-B. Les peptides de B.longum et de B.breve qui sont homologues à l’empreinte du rebord nord viral sur la séquence CAR, forment des liaisons d’hydrogène avec plusieurs résidus viraux dans la région du rebord nord du canyon, qui sont déjà décrits pour leur interaction avec le CAR.En conclusion, les protéines de LPAS bifidobactéries peuvent inhiber l'infection par le CV-B4 probablement par liaison aux acides aminés de la capside qui interagissent avec le CAR. / The present study aims at investigating the potential of bifidobacteria in protecting cells from Coxsackievirus B4 (CV-B4) infection. The bifidobacterial screening identified two out of five strains that protected HEp-2 cell viability when bifidobacteria were incubated with the viral particles prior inoculation. In contrast, no effect was shown by incubating HEp-2 cells with bifidobacteria prior CV-B4 inoculation. Cell-wall lipoproteins secreted by the selected strains (LpAs) were assayed for their anti-viral activity. The two LpAs exhibited anti-viral activity when they were incubated with the viral particles prior inoculation to HEp-2 cells. No effect was induced by incubating LpAs with HEp-2 cells prior CV-B4 inoculation. The recombinant LpAs derived-protein exhibited identical anti-viral activity. To identify the peptide sequences interacting with the virus particles, LpAs proteins were aligned with the peptide sequences of north canyon rim and puff footprint onto coxsackievirus and adenovirus receptor (CAR). The in silico molecular docking study using CV-B3 as template showed a low energy binding indicating a stable system for the selected peptides and consequently a likely binding interaction with CV-B. B.longum and B.breve peptides homologous to the viral north rim footprint onto CAR sequence formed hydrogen bonds with several viral residues in the north rim of the canyon, which were already predicted as interacting with CAR. In conclusion, proteins from bifidobacterial LpAs can inhibit the infection with CV-B4 likely through binding to the capsid aminoacids that interact with CAR.

Microorganisms have been known to play a major role in human health since early times. The ingestion of microorganisms as probiotics to restore and/or maintain health is a widely accepted and common practice. The challenge in industry is to ensure viability of probiotics until their ingestion to their site of action, the colon, for health benefits to be realised. Microencapsulation is one of the techniques used to protect probiotic bacteria and ensure viability. A method that does not involve the use of extreme temperatures and/or solvents which would otherwise adversely affect viable cells was developed and patented. The method is solventless and is based on complexation of Food and Drug Administration-approved polymers, poly (vinylpyrrolidone) and poly (vinylacetate-co-crotonic acid) in supercritical carbon dioxide. The use of this method of encapsulation was found to be suitable in target release in earlier studies. Microparticles produced were found to have pH-dependent swellability, protecting bioactives, in this case probiotic bifidobacteria, in acid (simulated gastric acid) and only releasing them in an alkaline environment (simulated intestinal fluid). Further studies were, however, needed to investigate the suitability of the microparticles for food and pharmaceutical applications. The current study therefore aimed to characterize these microparticles in terms of size range, distribution of bacteria within the microparticles, and particle size distribution. The average size of the Bifidobacterium lactis Bb12-encapsulating microparticles was found to be within the acceptable size in food applications. High encapsulation efficiency was obtained, with live bacteria distributed evenly within the microparticles, demonstrating the potential of the microparticles to deliver high numbers of probiotic cultures as required for this type of microorganisms to deliver purpoted benefits to the consumer. Probiotic products are normally kept under refrigerated storage, yet the viability of bacterial cells still decreases. An additional benefit of encapsulation within microparticles would be protection of the encapsulated probiotics from the detrimental factors to which the probiotic products are exposed during storage. In order to investigate this for the microparticles in this study, the shelf life of encapsulated B. lactis Bb 12 powder stored in glass vials was investigated. High temperatures were used for accelerated shelf life studies. Encapsulated B. lactis Bb 12 maintained the viable levels above the therapeutic minimum for the duration of the study (12 weeks), which was 7 weeks more than was the case with unencapsulated probiotic. Thus the microparticles provided protection to the probiotic cultures at temperatures much higher than those normally used for storage of probiotic products. These results further indicate the possibility for storage of the B. lactis Bb12 encapsulated in the tested microparticles, at ambient temperatures for at least two months, without drastic loss of culture viability. Research has recently focused on the development of probiotic foods other than dairy and dairy-based foods. This has been necessitated by increasing vegetarian lifestyle and concerns of allergenicity. A maize-based traditional fermented beverage, mageu, was investigated for use as a vehicle for probiotic delivery. Although no significant difference was noted between survival of encapsulated and unencapsulated probiotic was noted, pH decrease in mageu with encapsulated B. lactis Bb 12 was less than with unencapsulated cells. This suggested that encapsulation would ensure that metabolites produced by encapsulated probiotics, if any, would not negatively affect a product in which they are incorporated. Further studies may be needed for investigation of the effect of the encapsulating microparticles in traditional fermented non-dairy products, using more acid-sensitive probiotic strains as the test strain used in the current study is well-known for its inherent resistance to acidity. This study filled gaps in knowledge in terms of the characteristics of microparticles produced using supercritical technology. The main highlights of the research findings were that the microparticles were suitable for food applications, improved probiotic viability under nonrefrigerated temperatures, and delayed browning of the probiotic powder and minimized drop in pH of the fermented product containing the probiotic encapsulated within. The results showed that microparticles encapsulating B. lactis Bb 12 are appropriate to consumers in areas where refrigeration is absent. Furthermore, the study showed that mageu is a suitable alternative vehicle to dairy-based products, for delivery of probiotic B. lactis Bb 12. This possibility extends accessibility of probiotic products to consumers who do not take dairy products for various reasons. There is also a potential increase of probiotic products on the market. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

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The goal of this work was a study the influence of food and beverages on the viability and growth of probiotic bacteria. The influence of food and beverages was tested on monocultures of Lactobacillus acidophilus, Bifidobacterium breve and mixed culture of probiotic microorganisms. In the experimental part, probiotic cultures were incubated in selected foods and beverages. After that they were tested in a model conditions of digestive tract. In some probiotic cultures, growth of viable cells during incubation in the digestive tract was observed. The increase of probiotic cells was showed predominantly in foods that contained higher levels of sugars and fats or a suitable combination. Their increase was up to four times in some cases. Based on the results, mixed probiotic cultures aren’t surprisingly exhibited better survival and maintain sufficient amount of viable cells even during the digestive process. Moreover, probiotic microorganisms could be recommended to consumption during meals better than just with a beverage.

Irritable bowel syndrome (IBS) är en av våra vanligaste funktionella tarmsjukdomar med en prevalens på ca 10% värden över. Diagnosen ställs utifrån ROME-kriterierna vilka är ett symptombaserat diagnostiskt verktyg samt uteslutande av andra allvarliga tillstånd. Vidare kan IBS delas in i undergrupper beroende på avföringens konsistens mätt utifrån The Bristol stool form scale (BSFS). Patologin bakom sjukdomen är idag inte helt klarlagd men flera faktorer och mekanismer tros spela in. Däribland finns visceral hypersensitivitet och avvikande uttryck av smärtreglerande signalsubstanser, rubbningar i den gastrointestinala mikrobiotan, genetik, ökad inflammation, defekt mucosabarriär med ökad permeabilitet, psykosociala faktorer och kost. Behandling är främst inriktad på symptomlindring och att utbilda patienten i att hantera sjukdomen genom att undvika triggande faktorer som kan förvärra symptom. Probiotika är levande icke-patogena mikroorganismer som används i behandlande syfte. Några av de mest studerade släkterna av bakterier som används vid behandling av IBS är Lactobacillus och Bifidobacterium. Detta arbete syftade till att analysera effekten av probiotika i form av en kombination med dessa två släkten som symptomlindrande behandling vid IBS. Detta gjordes genom litteratursökning på databasen PubMed där fem artiklar filtrerades fram utifrån satta kriterier. Resultatet visade att probiotika innehållande Lactobaciullus och Bifidobacteirum har potential att förändra den gastrointestinala mikrobiotan. Man kunde också se att en ökad nivå Bifidobacteriumkorrelerade med sänkt symptomgrad och lägre nivå inflammatoriska markörer. Studierna visade också på sänkt tarmpermeabilitet efter intag av probiotika. Den generella symptombilden och allvarlighetsgraden av sjukdomen förbättrades och även flera specifika symptom. Trots det kunde en entydig och signifikant förbättring av livskvalitén inte fastställas. Dosen probiotika som användes var mellan 5-10 miljarder colony forming units (CFU) i alla utom en studie. Denna undersökte en betydligt högre dos om 52 miljarder CFU men gav trots det inte bättre resultat än övriga studier. Studiepopulationen var sammantaget enhetlig i de olika studierna och speglade också den vanligaste patientgruppen. Slutsatsen var att probiotika innehållande släkterna Bifidobacterium och Lactobacillus kan förbättra allvarlighetsgraden av IBS och ge en generell och specifik symptomlindring. / Irritable bowel syndrome (IBS) is one of the most common functional bowel disorders without any detectable biochemical or structural changes with a prevalence of about 10 % worldwide. The diagnosis is based on the ROME-criteria which is a symptom based diagnostic tool and also exclusion of other severe diseases. Further on, IBS can be divided into different subgroups based on stool texture evaluated by the Bristol stool form scale (BSFS).  The pathology behind IBS is not fully understood but several factors and mechanisms are thought to be involved. Amongst those are hypersensitivity in the gut and divergent pain transmission, disturbance in gut microbiota, genetics, increased inflammation, defective mucosal barrier function with increased permeability, psychosocial factors and diet. Treatment is mainly used to alleviate symptoms so as to help the patients to manage the disease by education and by avoiding the triggering factors that may exacerbate the symptoms.  Probiotics are living non-pathogen microorganisms, meaning they are not harmful, used to treat or favor the host. Some of the most studied genera of bacteria, both in general and used as treatment of IBS, are Lactobacillus and Bifidobacterium. These bacteria are often referred to as lactic acid bacteria due to their ability to metabolize specific carbohydrates and produce lactic acid.  This study aimed to investigate the effect of probiotics consisting of these two genera on symptom relief for patients with IBS. It was achieved by carrying out a literature review in the PubMed database. Five articles were found based on set criteria.  The results showed that probiotics based on Lactobacillus and Bifidobacterium have potential to alter the gut microbiota and that levels of Bifidobacterium corresponding to level of symptoms and inflammatory markers. Also, the intestinal permeability was lowered by probiotics administration. The overall symptoms and severity were decreased as well as several specific symptoms. Despite that, a significant increase in quality of life was not observed.  The dose used was between 5-10 billion colony forming units (CFU) in all but one study. This study used a much higher dose of 52 billion CFU and did also last a longer period of time but did not get better results than the other studies. The study population was in general similar in all studies and did also reflect the most common patient group based on prevalence.  In conclusion, probiotics consisting of Lactobacillus and Bifidobacterium can be used to improve severity of IBS and to relieve general and specific symptoms.

Self-injurious behavior (SIB) is a behavioral pathology seen in a small percentage of humans and non-human primates. In one previous study, macaques with SIB had more sleep disruption than controls, but observations were limited. Two studies were conducted: a baseline study to investigate nighttime activity in rhesus macaques (Macaca mulatta) displaying SIB and controls, and a probiotic study to assess probiotic Bifidobacterium infantis 35624 for high nighttime activity. Subjects were 13 rhesus macaques, 5 with SIB (3 females; 1 SIB). Videocapture of Nighttime Activity (VNRA) was developed to record in complete darkness. IR-receptive webcams were connected to a laptop running ISPYCONNECT, software which recorded movement. Subjects were observed during the entire lights-off period (8pm-7am). Measures included total movement time (TMT), movement in hour 1 (HR1) and hour 11 (HR11), and number of videos. In the baseline, SIB subjects had higher TMT (pBifidobacterium infantis 35624 had no effect on sleep disruption, and also that increased nighttime activity seems to be a persistent characteristic of SIB subjects. It is unknown if increased nighttime activity affects SIB subjects; it may result in elevated SIB, or the SIB pathology could result in sleep disruption.

Pour produire des ferments lactiques contenant des bifidobactéries par des cellules immobilisées, des fermentations ont été réalisées avec une culture pure de bifidobactéries ou en culture mixte avec une souche de lactocoques. Une méthode a été développée pour localiser et quantifier par immunofluorescence les deux souches dans des billes de gel. L'utilisation de cette méthode a permis d'observer une croissance bactérienne préférentielle à la périphérie des billes. La production continue de culture mixte a été étudiée avec un système composé d'un premier réacteur (R1) contenant les deux souches immobilisées séparément dans des billes de gel et d'un second réacteur (R2) opéré avec les cellules libres relarguées de R1. Une culture mixte concentrée de composition stable a été produite à 35ʻC dans l'effluent de R2. La redistribution des souches dans les billes fut observée en microscopie confocale. La résistance à différents stress des cellules libres produites dans l'effluent des deux réacteurs a augmenté avec le temps de fermentation.