A crescente preocupação com tecnologias sustentáveis e a procura de novos alimentos funcionais despertam o interesse para o desenvolvimento de novos produtos alimentícios que proporcionem, além da nutrição, benefícios à saúde do consumidor. Assim, esse trabalho visa propor o leite orgânico como potencial matéria-prima para a fabricação de leites fermentados probióticos. Para tanto, estudou-se o perfil tecnológico de cepas de bifidobactéria em cultura pura e em co-cultura com Streptococcus thermophilus em leites orgânico e convencional, analisando a composição química dos leites, determinando o perfil de acidificação de quatro cepas de Bifidobacterium animalis subsp. lactis em cultura pura e em co-cultura com Streptococcus thermophilus, verificando a contagem microbiológica das culturas probióticas e iniciadoras nos leites fermentados, examinando o perfil de ácidos graxos e o teor de ácido linoléico conjugado dos leites fermentados e determinando o perfil de textura dos leites fermentados. A maior velocidade de acidificação foi observada para as cepas B94 e BL04 em leite orgânico e para a cepa HN019, para ambos os tipos de leite. As contagens de todas as cepas de B. animalis subsp. lactis foram superiores a 8,58 log10 unidades formadoras de colônia (UFC).mL-1</SUP. O leite orgânico apresentou maiores teores de ferro e proteína, enquanto o leite convencional apresentou maiores teores de gordura e lactose. Os principais ácidos graxos foram pouco influenciados pelo tipo de leite e as maiores quantidades de ácido linoléico conjugado (65 % maior do que o controle) foram encontradas em leite orgânico fermentado com a cepa BB12 em co-cultura com S. thermophilus. Assim, verificou-se que o leite orgânico pode ser empregado como matéria-prima na fabricação de leites fermentados probióticos, agregando qualidade nutricional ao produto final. / The concern about sustainable technologies and demand for new functional foods arouses the interest for the development of new food products in addition to provide nutrition and health benefits to the consumer. Thus, this work aims to offer organic milk as a potential raw material for the manufacture of probiotic fermented milk. Therefore, studying the technological profile of strains of bifidobacteria in pure culture and in co-culture with Streptococcus thermophilus in organic and conventional milks, analyzing the chemical composition of milk; determining the profile of acidification of four strains of Bifidobacterium animalis subsp. lactis in pure culture and in co-culture with Streptococcus thermophilus; checking the counts of starter and probiotic cultures in fermented milks; examining the profile of fatty acids and conjugated linoleic acid content of fermented milks and the profile of texture of fermented milks. The highest rate of acidification profile was observed for the strains BL04 and B94 in organic milk and for the strain HN019 in both milks. The counts of all strains of B. animalis subsp. lactis were higher than 8.58 log10 colony forming units (CFU). mL-1. The organic milk had higher levels of iron and protein, whereas conventional milk had higher levels of fat and lactose. The main fatty acids were not influenced by the type of milk and higher amounts of conjugated linoleic acid (65% higher than the control) were found in organic fermented milk with the strain BB12 in co-culture with S. thermophilus. Thus, it was found that organic milk can be used as raw material in the manufacture of probiotic fermented milk, increasing nutritional quality to final product.

Probiotics are live microorganisms which when administered in adequate amounts confer one or more health benefits on the host. Different processing conditions, the acidic condition of the stomach and exposure to hydrolytic enzymes affect the viability and efficacy of probiotic organisms. This study investigated the protective effects of two biopolymers poly-gamma-glutamic acid (γ-PGA) and bacterial cellulose (BC) on probiotics during freeze drying and during exposure to simulated intestinal juices and bile salts. The antibacterial property of Bifidobacterium strains was also investigated against four pathogenic bacteria. γ-PGA, a naturally occurring biopolymer was produced by two bacteria (Bacillus subtilis ATCC 15245 and B. licheniformis ATCC 9945a) in GS and E media, γ-PGA yields of about 14.11g/l were achieved in shake flasks and molecular weight of up to 1620 k Da was recorded, γ-PGA production was scaled up in a fermenter with B. subtilis using GS medium. BC, an edible biopolymer was produced by Gluconacetobacter xylinus ATCC 23770 in HS medium and a modified HS (MHS) medium. A yield of about 1.37g/l was recorded and BC production with MHS medium was used for probiotic application. B. longum NCIMB 8809 B. breve NCIMB 8807 and B. animalis NCIMB 702716 showed the best antimicrobial properties against the investigated pathogens. Survival of Bifidobacterium strains was improved when protected with powdered BC (PBC) although γ-PGA offered better protection than PBC. Viability of B. longum NCIMB 8809, B. breve NCIMB 8807 and B. animalis NCIMB 702716 in simulated gastric juice (SGJ) and simulated intestinal juice with bile salts was improved when protected with 5% γ-PGA and 5% γ-PGA+PBC with a reduction of < 1 Log CFU/ml while a reduction of ≤2 Log CFU/ml was recorded in PBC protected cells. Protecting Bifidobacterium strains with γ-PGA, PBC or a novel γ-PGA + PBC combination is a promising method to deliver probiotic bacteria to the target site in order to confer their health benefits on the host.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O principal objetivo do trabalho foi realizar a sÃntese de oligossacarÃdeos prÃ-biÃticos por via enzimÃtica em sucos de cajÃ, jambo e siriguela e a secagem dos mesmos por spray drying. Para isso foram avaliadas as caracterÃsticas dos sucos e aplicado um planejamento fatorial compÃsito central 22 a fim de selecionar a melhor condiÃÃo de sÃntese. Os sucos foram preparados, corrigidas as concentraÃÃes de aÃÃcares e adicionados da enzima dextrana-sacarase, sendo conduzida a sÃntese atà o consumo total da sacarose. ApÃs a inativaÃÃo da enzima, por reduÃÃo do pH atà o valor original dos sucos, uma parte destes foi submetida a tratamento tÃrmico brando e armazenamento sob refrigeraÃÃo para que se pudesse avaliar a estabilidade microbiolÃgica, sendo a outra parte submetida à secagem, utilizando maltodextrina como adjuvante. Os sucos prÃ-biÃticos em pà obtidos foram caracterizados quanto à atividade de Ãgua, umidade, higroscopicidade, reconstituiÃÃo em Ãgua, cor e grau de caking. Foram realizadas anÃlises de vitamina C nos sucos recÃm-preparados e sintetizados, e atividade antioxidante nos sucos recÃm-preparados, sintetizados e reconstituÃdos. Os oligossacarÃdeos prÃ-biÃticos sintetizados nos sucos foram avaliados quanto à resistÃncia a hidrolises Ãcida e enzimÃtica comportando-se de maneira positiva, alÃm da capacidade de estimular o crescimento de bifidobactÃrias. Os sucos prÃ-biÃticos em pà apresentaram, de uma maneira geral, boas caracterÃsticas fÃsico-quÃmicas, tais como baixas umidades, higroscopicidades e atividades de Ãgua, curto tempo de reconstituiÃÃo em Ãgua, embora os sucos de cajà e jambo apresentarem elevada tendÃncia à aglomeraÃÃo. Os sucos tratados termicamente apresentaram altas contagens em bolores e leveduras, entretanto, nÃo foi detectada a presenÃa de coliformes. Durante a sÃntese enzimÃtica houve diminuiÃÃo da concentraÃÃo de vitamina C dos sucos enquanto que o processo de secagem interferiu no poder antioxidante dos mesmos. Os oligossacarÃdeos prÃ-biÃticos obtidos apresentaram resistÃncia à acidez e Ãs enzimas digestivas, propiciando ainda a multiplicaÃÃo de bactÃrias benÃficas ao ser humano (bifidobactÃrias). / The aim of this work was produce yellow mombin, red mombin and malay apple juices with prebiotics oligosaccharides and, after, subject to spray drying process. The juices were analyzed and it was applied a factorial planning to purpose the better synthesis conditions. The juices were prepared and corrected its sugar concentration, and aftrer it was added the dextran-sucrase enzyme. The synthesis was realized for 4 hours. The enzyme was inactivated for pH decreasing until the juice original pH. A part of the juice was subjected to microbiologic stability and the other part was subject to spray drying, using maltodextrin like dryer adjuvant. The powder of the prebiotic juices synthesized was characterized by moisture, water activity, higroscopicity, water reconstitution, color and caking degree. Vitamin C and antioxidants was analyzed. The oligosaccharides resistance of acid and enzymatic hydrolysis was analyzed, as well as the bifidobacterium growth. In general the physico-chemistry results were good, like low moistures, higroscopicity and water activity, short time of water reconstitution, meanwhile the yellow mombin end malay apple juices showed high caking degree. It wasnât find coliforms, but molds and yeasts were high. The vitamin C decreased during the synthesis, and the drying process interfered with antioxidant power. The prebiotics oligosaccharides obtained were resistant to acidity and the digestive enzymes, enabling the benefic bacterial multiplication.

Ice cream mix (12% fat, 11% milk solids nonfat, 12.5% sugar, and 4.5% corn syrup solids) was fermented with Lactobacillus acidophilus and Bifidobacterium bifidum. Half of the mix was heat treated at 82°C for 30 minutes and cooled to 40-41°C. The other half was warmed to 40-41°C and inoculated with the starter cultures. Both were made into ice cream and stored at -29°C. Survival of L. acidophilus and B. bifidum and of β-galactosidase activity were monitored during 17 weeks of frozen storage. Reinforced clostridial medium was used to enumerate culture bacteria. Colony counts, after fermentation, for both L. acidophilus and B. bifidum were about 5 x 108. The population of cultures decreased less than one log cycle after initial freezing. After 17 weeks storage the bacterial counts were 1 x 107 for B. bifidum and were 4 x 106 for L. acidophilus. During the same period, β-galactosidase activity decreased only 31%. Therefore, frozen fermented dairy products provide a good vehicle to supply β-galactosidase enzymes to people who are lactose maldigestors. Frozen fermented ice cream was prepared at four different pH's (5.0, 5.5, 6.0, 6.5) by blending fermented mix with unfermented mix and then was frozen to produce samples for sensory evaluation. All samples were strawberry flavored. These were then evaluated by 88 judges. The preferred pH, based on overall acceptance, was 5.5. A second sensory evaluation was conducted to compare heat-treated with non-heat-treated ice cream. There were no significant differences in appearance, texture, flavor, and overall acceptance between the two samples. Our study shows that ice cream is a suitable vehicle for delivering these beneficial microorganisms and enzymes to consumers.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23332号 / 生博第450号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 片山 高嶺, 教授 永尾 雅哉, 教授 上村 匡 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

A bifidobacteria mix (nine strains) was evaluated for its effect on the growth and survival of Escherichia coli O157:H7 (ATCC 43890) in 11% NFDM and MRS broth under the optimum growth conditions for E. coli O157:H7 growth (37¡ãC, aerobic). Preliminary experiments were conducted to obtain the growth curves of the 9 strains of bifidobacteria and E. coli O157:H7 and confirm the inhibitory effect of acidity on E. coli O157:H7 (pH of media adjusted to 3.8). Acidapted E. coli O157:H7 showed no difference in resistance toward bifidobacteria (P>0.05) when compared to the non-acid adapted one. Escherichia coli O157:H7 did not survive in the supernatant of the bifodbacteria mix collected after incubation (37¡ãC) with aerobic shaking (8 h). However, the pathogen was able to grow after the pH of the supernatant was adjusted to 6.50 (pH of fresh MRS broth). Results suggest that a high content of bifidobacteria has a strong inhibitory effect on E. coli O157:H7, in part due to the low pH. However, products from bifidobacteria may also exert inhibitory effects.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24672号 / 農博第2555号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5453(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 井上 善晴, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Bifidobacteria are the predominant members of the infant gut, colonize adults to a lesser extent, and are recognized as beneficial microbes. Various bifidobacterial species produce ��-aminobutryic acid (GABA), the chief inhibitory neurotransmitter in the mammalian central nervous system. It is postulated that in order to produce GABA, the bifidobacterial genome must contain the gadB and gadC genes which encode a glutamate decarboxylase and a glutamate/GABA antiporter, respectively. Once exported by GadC, GABA is absorbed and transported systemically throughout the host. We hypothesize that specific dietary oligosaccharides will modulate bifidobacterial production of GABA due to varying intracellular concentrations of glutamate. To test this, 33 bifidobacterial strains were screened for GABA production via reverse phase HPLC. Interestingly, 10 strains contained both gadB and gadC genes, but only 8 strains produced detectable GABA in vitro. To further elucidate the extrinsic factors influencing GABA production, strains were subjected to different dietary components. Specifically, lactose and the dietary oligosaccharide FOS were evaluated for the ability to promote biosynthesis of intracellular glutamate and thus potentially GABA. Understanding the relationship between diet, bifidobacterial physiology, and GABA production may inform dietary interventions to modulate this neurotransmitter in vivo.

Les bifidobactéries sont exposées à de nombreux stress, liés aux conditions environnementales rencontrées lors de la production, du stockage au froid, et pendant la digestion des laits fermentés. Afin d'améliorer leur survie, cette étude vise la compréhension des mécanismes de dégradation de l'état physiologique de différentes souches de Bifidobacterium soumises aux stress froid et acide et au stress gastro-intestinal simulé in vitro. Elle ambitionne également d'établir des relations entre la résistance aux différents stress et la teneur en acides gras membranaires et des laits biologiques et conventionnels. Les résultats montrent que l'activité acidifiante des bifidobactéries est souche-dépendante et qu'elle augmente lorsque les bactéries sont associées aux bactéries lactiques du yaourt, avec du lait biologique et lorsque la température d'incubation est fixée à 42°C au lieu de 37°C. La cultivabilité et la survie des souches ont été déterminées après fermentation, après stockage à 4°C pendant 7 à 28 jours, et pendant un processus de digestion simulé in-vitro dans un digesteur dynamique reproduisant le tractus gastro-intestinal. Ces caractéristiques sont améliorées dans les laits fermentés biologiques par rapport aux produits conventionnels, lorsque la fermentation est effectuée à 42°C jusqu'à pH 4,4, et lorsque les laits fermentés sont maintenus à 28°C pendant 12 heures avant d'être refroidi à 4°C. Ces procédures de fabrication spécifiques génèrent ainsi une adaptation physiologique des bifidobactéries aux stress. Pendant la digestion in-vitro, la cultivabilité des bifidobactéries se dégrade moins lorsque la fermentation se déroule en lait biologique plutôt qu'en lait conventionnel et, dans une moindre mesure, lorsque les procédures d'adaptation sont appliquées pendant la fabrication du lait fermenté. Ces résultats sont liés aux teneurs plus élevées en acides gras insaturés, en particulier en acides trans-vaccénique, linoléique conjugué et α-linolénique, qui caractérisent les produits biologiques. Ces profils d'acides gras particuliers aux laits biologiques permettent aux bifidobactéries de modifier leur composition en acides gras membranaires, en augmentant leur teneur en acides gras insaturés et en raccourcissant la longueur moyenne des chaînes d'acides gras saturés, adaptant ainsi leur fluidité membranaire. Lorsque les procédures de fabrication spécifiques sont mises en oeuvre pour induire une adaptation physiologique des bifidobactéries, la composition en acides gras des membranes se modifie différemment de ce qui est observé en lait biologique. Cette différence indique ainsi que d'autres mécanismes biologiques d'adaptation sont probablement impliqués, en particulier au niveau protéomique. Finalement, cette étude démontre que les modifications au niveau de la membrane contribuent à moduler la résistance aux stress technologique et gastro-intestinal de souches de Bifidobacterium. / Bifidobacteria are exposed to various stress, as a result of environmental conditions encountered during fermented milk production, cold storage and during digestion of the products inside gastrointestinal tract. In order to improve their survival, this study aimed at understanding the degradation mechanisms of the physiological state of various Bifidobacterium strains when exposed to cold, acid and in vitro simulated gastrointestinal stress. It also intended to establish relationships between stress resistance and milk and membrane fatty acids contents, in organic and conventional milks. The results showed that acidification activity of bifidobacteria was strain-dependent and increased when bifidobacteria were associated to yogurt cultures, when organic milk was used and when incubation temperature was set at 42°C instead of 37°C. Cultivability and survival of the Bifidobacterium strains were determined after fermentation, after storage at 4°C for 7 to 28 days, and during in-vitro digestion that was simulated in a dynamic gastrointestinal tract model. These characteristics were improved in organic fermented milks as compared to conventional products, when fermentation was performed at 42°C until pH 4.4, and when the fermented milks were kept at 28°C for 12 hours before being cooled to 4°C. These specific manufacture procedures thus generated physiological adaptation of the bifidobacteria to the stress. During in-vitro digestion, cultivability of bifidobacteria was less deteriorated when they were grown in organic instead of conventional milk, and to a less extent, when the adaptation procedures were applied during fermented milk manufacture. These results were related to the higher unsaturated fatty acids content, including trans-vaccenic, conjugated linoleic and α-linoleic acids that characterize organic products. These particular fatty acids profiles of organic milks allowed bifidobacteria to modify their membrane fatty acids composition, by increasing their unsaturated fatty acids contents and by shortening the length of medium chain saturated fatty acids, thus adapting their membrane fluidity. When specific manufacture procedures were carried out to trigger physiological adaptation of the bifidobacteria, membrane fatty acid composition changed différently from what is observed in organic milk. This difference indicates that other biological adaptation mechanisms are probably involved, especially at the proteomic level. Finally, this study demonstrated that modifications at membrane level contribute to modulate resistance against technology and gastro-intestinal stress of Bifidobacterium strains to better withstand technological and gastro-intestinal stress.

Nowadays, probiotic lactic acid bacteria (LAB) and bifidobacteria are plentifully exploited in food processing industry. LAB and bifidobacteria are important part of microflora of gastro intestinal tract (GIT). Probiotics (most often just lactobacilli and bifidobakteria) can be supplied to the GIT of the organism like food complements. Species identification is therefore very important. New methods of identification of LAB and bifidobacteria are based on analysis of DNA. Mostly exploited method is polymerase chain reaction (PCR). In my diploma work, genus and species specific PCRs were used for identification of different species of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of six food supplements (Zenflo, Linex Forte, Probian, Nutra Bona, GS lactobacillus Forte, Pangamin Bifi plus). Total DNA was isolated from crude lysates of cells present in tablets by magnetic particles coated by carboxyl groups . The preparation of cell lysates was optimalised. Different amounts of lysozyme (3 mg/ml, 10 mg/ml), time of incubation at laboratory temperature (1,5 hour, 3 hour) and time of incubation with SDS and proteinase K at 55 °C (1 hour, 3 hour, over all night) were tested. Isolated DNA was quantified and checked in PCR. Primers specific for genus Lactobacillus and Bifidobacterium and for species Lb. acidophilus, Lb. casei, Lb. rhamnosus and Lb. plantarum and B. animalis, B. bifidum, B. infantis, B. longum were used, respectively. All identified bacteria were in accord with the data declared by producer in 3 food supplements (Zenflo, Linex Forte and Pangamin Bifi Plus). The genus indentification was in accord with declaration of producer in other food products only (Probian, Nutra Bona, GS Laktobacily Forte).