Rozprawy doktorskie na temat „Microbial exopolysaccharides”

Paenibacillus velaei, sp. nov. is a soil bacterium capable of producing an unusually large amount of exopolysaccharide (EPS). The EPS contains glucose, mannose, galactose and fucose in a molar ratio of 4:2:1:1. The molecular weight of the EPS is higher than 2x106. The viscosity of 1% EPS is 1300 cP when measured at a shear rate of 1 sec-1. Physiological parameters for optimal production of the EPS were studied and it was found that 1.4 g dry weight per 1 l of medium was produced when the bacteria were grown at 30EC and the pH adjusted at 7± 0.2 in a medium containing glucose as the carbon source. Growing the bacteria on different carbon sources did not alter the quantity or the composition of the EPS produced. No toxicity effects were observed in mice or rats when EPS was administered in amounts ranging from 20 to 200 mg per kg body weight. The data obtained from physical, chemical and biological properties suggest that the EPS may be employed in several industrial and environmental applications. It is an excellent emulsifier, it holds 100 times its own weight in water, it is not toxic, and it can be used to remove mercury, cadmium and lead from aqueous solutions.

Lactobacilli are intestinal bacteria with known immunomodulatory competence. Numerous strains of this genus have been implicated in both the prevention and treatment of intestinal inflammation as well as in maintenance of immunological homeostasis. The frequent inclusion of lactobacilli in probiotic products attests to this ability. These lactic acid bacteria colonise the murine forestomach and burgeon in other environments similarly rich in carbohydrate-containing substrates. Accordingly, lactobacilli may utilise fermentable carbohydrates to synthesise exopolysaccharides (EPS). These polymers are secreted into the cellular milieu and, while the ecological function of EPS is yet to be defined, evidence points towards a protective role. This function may include bacterial protection from immunological attack, via EPS recognition by immune cells, resulting in modulation of immunological activity. Dendritic cells (DC) are potent antigen presenting cells, providing an essential link between the innate and adaptive arms of the intestinal immune system. DC efficiently sample intestinal antigens and present peptides to cognate naive CD4⁺ T cells in secondary lymphatic tissue. Under the influence of secreted cytokines, DC direct the differentiation of naive CD4⁺ T cells and therefore, instruct the resultant immune response. Anti-inflammatory Th2 and regulatory T cells can down-regulate the destructive Th1 pro-inflammatory effects associated with inflammatory bowel disease (IBD). As such, bioactives with the aptitude to direct DC activity and T cell differentiation have the potential to prevent or reduce intestinal inflammation. Therefore, this study aimed to determine whether heat-killed EPS-producing strains of lactobacilli, and their secreted EPS, exert an immunomodulatory effect on the murine gut which may down-regulate the immune reactions associated with IBD. Lactobacilli were screened for their ability to produce EPS when grown in the presence of glucose, sucrose or lactose. Heat-killed EPS-producing strains were then used to stimulate bone marrow-derived DC (BMDC) and the resultant cytokine profile was analysed. Nine Lactobacillus strains were found to produce EPS when grown in the presence of sucrose. Of these, L. reuteri 100-23 and L. johnsonii 100-33 exhibited potential anti-inflammatory effects. Therefore, these strains, as well as L. johnsonii 100-5 and L. johnsonii #21, with relatively weak BMDC stimulatory effect, were selected for further investigation. EPS of the potentially anti-inflammatory strain L. reuteri 100-23 was analysed. This sample contained approximately 85% carbohydrate and was composed of a (2[to]6)-β-fructofuranan (levan) and a mannan. The fructan, with an estimated molecular weight of 7 kDa, comprised at least 50% of the EPS, while the mannan made up at least 22%. The mannan component was likely linked to a protein and may have originated from the culture medium. The immunostimulatory capacity of heat-killed Lactobacillus bacterial cells and their EPS was determined in vitro. Firstly, the effect of lactobacilli and EPS on BMDC cytokine secretion, particularly levels of anti-inflammatory IL-10 and pro-inflammatory IL-12, as well as the expression of cell surface activation markers, was determined. L. reuteri 100-23 stimulated relatively high IL-10 secretion but low IL-12, while L. johnsonii 100-33-stimulated BMDC produced elevated levels of both IL-10 and IL-12. All bacterial cells up-regulated co-stimulatory molecules CD40 and CD80 on BMDC. The effect of these stimulated BMDC on T cell proliferation and cytokine production was then assessed, employing the ovalbuminDO11.10 T cell model. L. reuteri 100-23-stimulated BMDC down-regulated T cell production of the proliferation-stimulating cytokine, IL-2, up-regulated regulatory TGF-β secretion, but did not affect pro-inflammatory IFN-γ levels. The EPS of all strains did not stimulate significant BMDC cytokine production and failed to alter BMDC activation marker expression. However, BMDC stimulated with L. reuteri 100-23 and L. johnsonii 100-33 EPS significantly enhanced T cell IL-2 secretion, but did not alter TGF-β or IFN-γ levels. The effect of in vivo L. reuteri 100-23 and EPS intestinal stimulation on the reactivity of immune cells was subsequently investigated. Mesenteric lymph node (MLN) cells and splenic T cells from reconstituted Lactobacillus-free mice fed stimulant or PBS on two occasions were co-cultured with stimulated or unstimulated donor CD11c⁺ splenic DC ex vivo. Cellular proliferation as well as TGF-β and IFN-γ secretion was analysed, and IL-10 neutralisation assays were carried out to ascertain the involvement of this cytokine. Primary exposure of MLN cells to L. reuteri 100-23 resulted in suppressed cell proliferation in the presence of enhanced TGF-β levels, which may have also involved IL-10. Primed splenic T cells exhibited increased proliferation in the presence of elevated TGF-β levels following re-exposure to L. reuteri 100-23, and IL-10 may be involved in limiting this proliferation. L. reuteri 100-23 EPS did not alter MLN cell proliferation, possibly due to the suppressive activity of IL-10, but did enhance that of naive and primed splenic T cells. The effect of ingested L. reuteri 100-23 and EPS on intestinal sIgA concentration was assessed by quantifying IgA levels in the faecal supernatant of RLF mice previously ingesting L. reuteri 100-23 and EPS. L. reuteri 100-23 EPS-fed female mice exhibited significantly elevated levels of sIgA, while heat-killed bacteria did not affect antibody levels. The present study demonstrated that oral administration of heat-killed L. reuteri 100-23 and EPS exerts immunomodulatory effects on the murine intestine. These bioactives may promote a suppressive environment by conditioning DC to secrete a cytokine profile conducive to regulatory T cell induction and memory generation. Additionally, mucosal protection may be favoured by the stimulation of elevated sIgA levels. Therefore, a therapeutic composite is possibly obtained to preserve the intestinal barrier by defending against pathogen-induced injury and buffering inflammatory events. In these ways, L. reuteri 100-23 and EPS may confer long-lasting protection against, and down-regulate the immune reactions associated with, IBD.

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CAPES
Os polissacarídeos microbianos estão sendo muito utilizados atualmente por causa das vantagens em relação aos provenientes de outras fontes. Muitos são sintetizados por bactérias pertencentes à família Sphingomonadaceae como gelana, ramsana, welana, diutana, entre outras. Apesar da quantidade de polissacarídeos existentes, a descoberta de novos polissacarídeos microbianos é importante, tendo em vista a sua vasta aplicabilidade industrial, como espessantes, emulsificantes, estabilizantes e quelantes. Além disso, há a possibilidade de propriedades mais vantajosas e maior produção bacteriana. Este trabalho teve como objetivo selecionar linhagens bacterianas nativas de ambiente marinho produtoras de exopolissacarídeos e caracterizá-los. Neste contexto, a otimização da composição dos meios de cultivo e condições de processo podem modificar a produção, com possibilidade de aplicação industrial. Quatro bactérias foram selecionadas a partir da Coleção de Cultura Microbiana do Instituto de Ciências da Saúde pela resistência ao meio ágar nutriente contendo o antibiótico estreptomicina nas concentrações 100 e 200 μg.mL-1, sendo posteriormente identificadas por análise molecular como pertencentes aos gêneros Sphingomonas sp., Sphingobium sp. e Bacillus sp. A produção dos polímeros sintetizados por essas bactérias foi realizada em meio de cultivo, com alteração da fonte de carbono (sacarose ou glicerina bruta). A quantidade dos exopolissacarídeos sintetizados pelas bactérias pertencentes aos gêneros Sphingomonas sp. e Bacillus sp foi de 0,2 g.L-1 independente da fonte de carbono utilizada. O polímero produzido por Sphingobium sp. foi de 0,1 g.L-1 no meio contendo sacarose e 0,2 g.L-1 no meio com glicerina bruta. A CCMICS SB 22 não produziu exopolissacarídeo no meio contendo sacarose, enquanto que com a glicerina bruta foi de 0,2 g.L-1. As viscosidades dos exopolissacarídeos produzidos pelas quatro linhagens estudadas não apresentaram diferença entre si. A massa molecular do exopolissacarídeo produzido por Sphingobium sp. foi de 1,13 x 103 Daltons. Os outros polímeros não tiveram a massa molecular determinada por não apresentarem solubilidade em água.
The microbial polysaccharides are currently used being much because of advantages over from other sources. Most of those which are being studied are synthesized by bacteria of Sphingomonadaceae family, like gelan, rhamsan, welan, diutan, among others. Despite the amount of existing polysaccharides, the discovery of new polysaccharides is important, in view of its wide industrial applicability, such as thickeners, emulsifiers, stabilizers, and binders. Furthermore, there is the possibility of further advantageous properties and increased bacterial production. This work aimed to select native bacterial strains of exopolysaccharides-producing marine environment and characterize them. In this context, optimization of the composition of culture media and process conditions may change the production, with the possibility of industrial application. Four bacteria were selected from the Microbial Culture Collection of Sciences Institute of Health the resistance to the nutrient agar containing the antibiotic streptomycin in concentrations 100 and 200 μg.mL-1, subsequently identified by molecular analysis as belonging to the Sphingomonas sp., Sphingobium sp. and Bacillus sp. genres. The production of polymers synthesized by those bacteria was held in the culture medium, by changing the carbon source (sucrose or crude glycerin). The quantity of synthesized exopolysaccharides by the bacteria belonging to the Sphingomonas sp. and Bacillus sp genres was 0,2 g.L-1 regardless of the carbon source used. The polymer produced by Sphingobium sp. was 0,1 g.L-1 in the medium containing sucrose and 0,2 g.L-1 in the medium with crude glycerin. The CCMICS SB 22 produced no exopolysaccharide in the medium containing sucrose, while with crude glycerin was 0,2 g.L-1. The viscosities of exopolysaccharides produced by the four strains studied did not differ among themselves. The molecular mass of the exopolysaccharide produced by Sphingobium sp. was 1,13 x 10³ Daltons. The others polymers did not have the molecular mass determined for not showing solubility in water.

This thesis focused on the study of the influence of different exopolysaccharide types produced by two strains of Streptococcus thermophilus on the physical properties of fermented milk. First, the fermentation factors affecting EPS production were studied to ascertain required carbon source and environmental conditions which would support their production. Higher fermentation temperature (42°C) resulted in a greater cell growth and EPS production. EPS production was growth associated in glucose or lactose-containing M17 medium. The examined strains appeared to be able to utilize galactose for the EPS assembly and produced comparable amounts of EPS, albeit restrictive cell growth. The EPS production of the two strains was comparable, ranging from ~100 to ~600 mg/L. Secondly, the EPS were rheologically characterized to show their resistance to deformation. Influence of temperature, pH and concentration on the flow behaviour of these EPS was also assessed. Under acidic conditions, capsularropy EPS was less responsive to temperature with a higher zero shear viscosity ηo (14.36 to 150.82 mPa s) than capsular EPS (93.72 to 9.24 mPa s), and slightly higher relaxation time τ (0.43 to 15.82 s for capsular-ropy EPS and 0.72 to 9.36 s for capsular EPS). The opposite behavior was observed under neutral pH. EPS concentration did not give significant effect (P>0.05) on ηo and τ. The second study examined the effects of types of EPS on yoghurt texture under selected conditions. Fermented milk made using capsular-ropy EPS showed greater resistance to flow with less solid-like behaviour. It also had greater water holding capacity although the milk gel was less compact and brittle compared to fermented milk with capsular EPS. The EPS production in milk during fermentation between the two strains was comparable with maximum concentration was 840 plus/minus 47.5 mg EPS/kg fermented milk. Syneresis was lower in fermented milk incubated in low temperature, was ranging from 4.1-2.4 g/100 g fermented milk with capsular-ropy-EPS, and 10.9-26.6 g/100 g in fermented milk with capsular EPS. G’ was 23.8-365.1 Pa and 57.6-1040 Pa for fermented milk with capsular ropy and capsular EPS, respectively. The third study examined the involvement of EPS in the texture creation of fermented milk supplemented with calcium and/or sucrose, or calcium and whey proteins. Calcium addition to milk base resulted in increased acidity and greater syneresis (~20-30 g/100 g in fermented milk with capsular-ropy EPS and ~30-50 g/100 g in fermented milk with capsular EPS) and thixotropy of fermented milk, as compared to fermented milk without added calcium. Sucrose affected the parameters in opposite manner. EPS production did not differ from that of the control fermented milk. Storage modulus (G’) was 96-230.4 Pa, and 502.8-1143.5 for fermented milk with capsular ropy and capsular EPS, respectively. The effect of heat-untreated whey protein isolate or whey protein concentrate on calcium-fortified fermented milk was studied using capsular ropy EPS producer. Result showed that combined effect of both supplement was detrimental to texture of fermented milk to make it resemble that of drinking yoghurt. Syneresis was up to ~50 g/100 g, while G’ was only around 4 mPa. The next experiment studied the effect of heat-treated whey protein isolate addition on fermented milk texture. Results showed that heat-treatment applied to added whey protein preserved the G’ and syneresis with the values close to those of normal fermented milk. However, at high concentration of added heat-treated whey protein (whey protein:casein 3:1), the texture became very hard with 0 m2 permeability. Gelation was started very early in fermented milk added with heatdenatured whey protein. Whey protein addition induced the beginning of gelation. Supplemented fermented milk made using capsular-ropy EPS producer consistently showed lower G’, lower syneresis, and more shear-resistant compared to that made using capsular EPS. In conclusion, capsular ropy EPS, both in dispersion and in fermented milk with or without different supplementation, exhibited less solid-like properties and more shear-resistant behavior compared to capsular EPS.

This thesis focused on the study of the influence of different exopolysaccharide types produced by two strains of Streptococcus thermophilus on the physical properties of fermented milk. First, the fermentation factors affecting EPS production were studied to ascertain required carbon source and environmental conditions which would support their production. Higher fermentation temperature (42°C) resulted in a greater cell growth and EPS production. EPS production was growth associated in glucose or lactose-containing M17 medium. The examined strains appeared to be able to utilize galactose for the EPS assembly and produced comparable amounts of EPS, albeit restrictive cell growth. The EPS production of the two strains was comparable, ranging from ~100 to ~600 mg/L. Secondly, the EPS were rheologically characterized to show their resistance to deformation. Influence of temperature, pH and concentration on the flow behaviour of these EPS was also assessed. Under acidic conditions, capsularropy EPS was less responsive to temperature with a higher zero shear viscosity ηo (14.36 to 150.82 mPa s) than capsular EPS (93.72 to 9.24 mPa s), and slightly higher relaxation time τ (0.43 to 15.82 s for capsular-ropy EPS and 0.72 to 9.36 s for capsular EPS). The opposite behavior was observed under neutral pH. EPS concentration did not give significant effect (P>0.05) on ηo and τ. The second study examined the effects of types of EPS on yoghurt texture under selected conditions. Fermented milk made using capsular-ropy EPS showed greater resistance to flow with less solid-like behaviour. It also had greater water holding capacity although the milk gel was less compact and brittle compared to fermented milk with capsular EPS. The EPS production in milk during fermentation between the two strains was comparable with maximum concentration was 840 plus/minus 47.5 mg EPS/kg fermented milk. Syneresis was lower in fermented milk incubated in low temperature, was ranging from 4.1-2.4 g/100 g fermented milk with capsular-ropy-EPS, and 10.9-26.6 g/100 g in fermented milk with capsular EPS. G’ was 23.8-365.1 Pa and 57.6-1040 Pa for fermented milk with capsular ropy and capsular EPS, respectively. The third study examined the involvement of EPS in the texture creation of fermented milk supplemented with calcium and/or sucrose, or calcium and whey proteins. Calcium addition to milk base resulted in increased acidity and greater syneresis (~20-30 g/100 g in fermented milk with capsular-ropy EPS and ~30-50 g/100 g in fermented milk with capsular EPS) and thixotropy of fermented milk, as compared to fermented milk without added calcium. Sucrose affected the parameters in opposite manner. EPS production did not differ from that of the control fermented milk. Storage modulus (G’) was 96-230.4 Pa, and 502.8-1143.5 for fermented milk with capsular ropy and capsular EPS, respectively. The effect of heat-untreated whey protein isolate or whey protein concentrate on calcium-fortified fermented milk was studied using capsular ropy EPS producer. Result showed that combined effect of both supplement was detrimental to texture of fermented milk to make it resemble that of drinking yoghurt. Syneresis was up to ~50 g/100 g, while G’ was only around 4 mPa. The next experiment studied the effect of heat-treated whey protein isolate addition on fermented milk texture. Results showed that heat-treatment applied to added whey protein preserved the G’ and syneresis with the values close to those of normal fermented milk. However, at high concentration of added heat-treated whey protein (whey protein:casein 3:1), the texture became very hard with 0 m2 permeability. Gelation was started very early in fermented milk added with heatdenatured whey protein. Whey protein addition induced the beginning of gelation. Supplemented fermented milk made using capsular-ropy EPS producer consistently showed lower G’, lower syneresis, and more shear-resistant compared to that made using capsular EPS. In conclusion, capsular ropy EPS, both in dispersion and in fermented milk with or without different supplementation, exhibited less solid-like properties and more shear-resistant behavior compared to capsular EPS.

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia
The present work is focused on the production and characterization of oil in water emulsions stabilized with a bacterial exopolyssacharide (EPS), named FucoPol, produced by the bacterium Enterobacter A47 using glycerol as carbon source. The stabilizing ability of FucoPol was studied using aqueous biopolymer solutions with concentrations of 0.5%, 1.0% and 1.5% w/w, and sunflower oil, in ratios oil/water (O:W): 20:80, 40:60, 60:40 and 80:20. It was observed that the majority of the emulsions, except the proportions 80:20, showed no phase separation after 24 hours of maturation at 4 ºC. Emulsions had a shear thinning behavior, and it was observed that, for the same oil/water ratio, the apparent viscosity increased with increasing of FucoPol’s concentration in the aqueous phase. It was also found that either the apparent viscosity or viscoelastic properties remained quite similar over 72h, indicating the presence of stable emulsions during this period of time. The effect of FucoPol on the production of low-fat emulsions was also studied using pea protein (3% w/w) as emulsifier. It was studied the effect of FucoPol and oil concentrations on the characteristics of the emulsions obtained, keeping constant the emulsifier concentration. It was observed that for oil concentrations between 20% and 40% w/w, there’s a significant increase in viscosity with increasing of FucoPol’s concentration, but for oil contents between 40% and 60% w/w, no significant influence was observed. Still, for the whole range of oil concentrations tested it was observed that an increase in FucoPol concentration allows to produce emulsions with a stronger internal structure. Therefore, it was concluded that the adding of this biopolymer allows to produce emulsions with a fat content below 60%.

Antarctic marine bacteria isolated from sea ice and Southern Ocean particulate material were screened for exopolysaccharide (EPS) production. Ten strains were characterized using phenotypic (morphology), chemotaxonomic (whole cell fatty acid profiles) and phylogenetic (16S rDNA sequencing) techniques. These isolates were representatives of four genera including Pseudoalteromonas, Shewanella, Polaribacter and Flavobacterium, with one strain constituting a new bacterial genus in the family Flavobacteriaceae. After further phenotypic characterisation, this strain was given the name Olleya marilimosa, gen. nov., sp. nov. The ten strains were grown in batch culture and the EPS extracted, purified and partially characterized. Crude chemical, monosaccharide and molecular weight determinations showed that the EPS were diverse, even among closely related isolates. All EPS contained uronic acids to varying degrees and some also contained sulfate groups. Two EPS showed the presence of acetyl groups, with pyruvate present in at least one polysaccharide. The bacteria belong to phylogenetic groups that are dominant in sea ice and Southern Ocean particulate material according to previous studies that used culture dependent and independent techniques. These isolates were psychrotolerant, grew between 2 to 25°C and had growth optima at approximately 20°C. Growth and EPS production of one isolate belonging to the genus Pseudoalteromonas was examined at three temperatures: -2°C, 10°C and 20°C. EPS yield at -2°C and 10°C was thirty-fold higher than at 20°C. The EPS showed higher levels of uronic acids at lower temperature. The metal binding ability of a high molecular weight, highly viscous EPS produced by one sea ice bacterium was examined. High affinities for cadmium and copper were observed at the low concentration of EPS used. These results are a first step in assessing the ability of EPS produced by Antarctic marine bacteria to chelate dissolved trace metal such as iron, which are essential for growth and are limiting primary production in the Southern Ocean. The examination of EPS production by Antarctic marine bacteria provides new evidence that these biopolymers are abundant and diverse. Partial structural elucidation reveals important structure-function relationships. EPS such as those examined in this study may have a cryoprotective role or may impact on the availability of important trace metals. These findings point to the wider ecological role of EPS within the Antarctic marine environment. This study also provides incentive for further investigation into commercial usefulness of these biopolymers.

Lactococcus lactis subsp. cremoris Ropy 352 and L. lactis subsp. cremoris Hollandicus produce an exopolysaccharide (EPS) that imparts commercially desirable textural and rheological properties to fermented milk products. This ropy phenotype is expressed under specific environmental conditions. A mucoid EPS phenotype, also expressed under specific environmental conditions, but not involved in the fermentation of ropy milk was identified. The two EPS phenotypes can be expressed individually or concurrently. Genetic regulators involved in expression of the EPS phenotypes were sought. DNA probes and polyclonal antiserum specific to two regulators of EPS in Escherichia coli, Lon protease and RcsA protein, were used to probe ropy and non-ropy strains of L. lactis. The two ropy strains of L. lactis subsp. cremoris, Ropy 352 and Hollandicus, expressed significantly less of the Lon protein than non-ropy strains. Southern and Western blot analysis was extended to a number of Gram negative and Gram positive bacteria. All of the Gram negative bacteria probed contained DNA sequences that hybridized to the Ion and rcsA gene probes, and all of these bacteria has at least one protein that reacted with antiserum to E. coli Lon and RcsA proteins. Two of the Gram positive bacteria contained DNA sequences that hybridized to the E. coli rcsA probe. None of the other Gram positive organisms contained DNA sequences that hybridized to the rcsA or the Ion probes. However, all the Gram positive bacteria contained one high molecular weight protein that reacted with Lon antiserum. In addition, Streptococcus salivarius expressed a protein that reacted with RcsA antiserum. In the course of this study, a second RcsA protein was identified in E. coll. The two RcsA proteins are expressed from one rcsA gene. One RcsA protein is not the proteolytic product of the other RcsA protein. Limited peptide digest profiles of each RcsA protein reveals almost identical peptides indicating the two proteins share a high degree of homology but are not identical. Ferguson plot analysis strongly suggests that the two RcsA proteins differ by size not by charge. Neither RcsA protein can be detected in cells mutant for Ion and rcsB.
Graduation date: 1997

The genes coding for the expression of a ropy exopolysaccharide responsible for commercially desirable textural and rhealogical traits in fermented milk products by a natural lactococcal ropy isolate were sought. Using a transposon mutagenesis vector, pGh9:ISS1, three mutants lacking expression of the ropy exopolysaccharide were isolated. One of the mutants was chosen for further characterization. Using a Southern hybridization analysis, the interrupted gene was localized to the chromosome. The non-ropy mutant was further characterized and shown to be unable to produce ropy exopolysaccharide in fermented milk. A 2006 by fragment of the interrupted gene was sequenced. The DNA sequence over a short region showed homology to sugar transfer enzymes found in exopolysaccharide biosynthesis pathways. The DNA sequence was translated into its predicted amino acid sequences and two partial open reading frames of 236 and 338 amino acid residues in length were identified. These open reading frames were found to exhibit identity to glycosyltransferases present in exopolysaccharide biosynthesis pathways in other bacteria.
Graduation date: 1999

The general objective of this research was to develop a low-fat probiotic yogurt using fat replacers and EPS producing strains of Streptococcus thermophilus along with probiotics and to establish its antihypertensive property in vitro and in vivo. The specific aims of this project were: a) to select suitable strains of S. thermophilus and L. delbrueckii ssp. bulgaricus and of probiotics (L. casei, L. acidophilus and Bifidobacterium longum) based on their proteolytic, ACE-inhibitory and α-glucosidase inhibitory activities; b) to study the influence of a fibre-based fat replacer (Raftiline HP) and a protein-based fat replacer (Versagel) on the growth, proteolytic, ACE-inhibitory and α-glucosidase inhibitory activities of selected LAB; c) to select a suitable level of incorporation of the fat replacers in low-fat yogurt to obtain desired textural characteristics and to study the influence of these additions on the growth, proteolytic and ACE-inhibitory activities of S. thermophilus and L. delbrueckii ssp. bulgaricus during storage; d) to study the protective role of EPS, with and without the selected fat replacer and/or probiotics on growth and survival of S. thermophilus and L. delbrueckii ssp. bulgaricus and probiotics; yield of EPS, proteolytic and ACE-inhibitory activities; and spontaneous whey separation and textural properties of low-fat yogurt during storage; and e) to study the anti-hypertensive and hypocholesterolemic effects of the low-fat yogurt developed in this study by conducting a feeding trial using rats as a model.

The overall objective of this study was to investigate effects of variation in casein to whey protein ratios and types of exopolysaccharide producing starter cultures on physical properties of yoghurts made at 9% and 14% total solids during 28 days of storage at 4°C. There are three main parts of this research: a preliminary study, a comparative study of three methods for determination of syneresis and the studies on physical properties of yoghurts made with various casein to whey protein ratios using non-exopolysaccharide or exopolysaccharide-producing starter cultures.

This PhD work proposes the use of two microbial exopolysaccharides, GalactoPol and FucoPol, for the development of membranes to be applied in relevant dehydration processes in industry. The starting material is obtained from a low cost, abundant carbon source, the glycerol produced as a by-product from the biodiesel industry, which represents a key economic advantage. A special attention was given to the development of membranes with enhanced mechanical and transport properties, and superior water selectivity for application in dehydration processes by pervaporation and vapour permeation. The approaches selected for designing target membrane properties included polymer cross-linking and the development of hybrid membranes using the sol-gel method. Firstly, the membranes were modified using different crosslinking agents: trichloroacetic acid for GalactoPol and genipin for Fucopol. Different crosslinking protocols were studied in order to evaluate their potential use for ethanol dehydration by pervaporation. These membranes were characterised in terms of their morphological structure, resistance to solvents and mechanical properties. They showed high water affinity, good chemical resistance towards organic solvents and adequate mechanical properties. Pervaporation experiments were performed using both types of membranes for different water concentration in the feed stream (5.0 to 10.0 wt.%) at a constant temperature of 30 ºC. In addition, the impact of the exopolysaccharide purification protocol – by dialysis and by-dia-ultrafiltration – on the properties and transport performance of composite FucoPol membranes for pervaporation was also studied. Significant differences were found in the swelling behaviour and transport selectivity depending on the purification method. The shear stress imposed during purification by the dia-ultrafiltration method led to a disintegration of polysaccharide aggregates, and, as consequence, denser membranes were obtained, affecting the transport selectivity. The membranes developed, in particular, the composite GalactoPol-PES and dia-ultrafiltrated FucoPol-PES membrane, exhibited a high potential for ethanol dehydration, since a water/ethanol selectivity of 134 and 143, respectively, at 10.0 wt.% water in ethanol was achieved. Although they showed excellent affinity for water, they become progressively unstable in aqueous solutions. Thus, novel hybrid FucoPol membranes were developed combining the best properties of the inorganic network with the selectivity of the microbial polysaccharides, to be applied in pervaporation processes for ethanol dehydration, as well as, in gas dehydration. The hybrid membranes were prepared by incorporation of a SiO2 network homogeneously dispersed by a sol-gel method using (3-Glycidyloxypropyl) trimethoxysilane (GPTMS) as a crosslinker silica precursor. These membranes were structurally, mechanically and thermally characterised. They presented a dense and homogeneous structure, resistant to deformation, a glass transition temperature (Tg) of 43 ºC and a thermal decomposition between 240-251 ºC. Hybrid FucoPol membranes were successfully applied in ethanol dehydration, with higher selectivity values than commercial membranes PERVAP® 4101. However, they lost their stability in contact with solutions of 10.0 wt.% water in ethanol after three days of operation. In contrast, when these membranes were applied for N2 dehydration, they were stable, showed reproducible results and extremely high water selectivities. Permeation of water vapour and pure gases (CO2, CH4 and N2), at different conditions of gas humidity content was monitored by mass spectrometry. Gas mixtures containing 20 vol.% CO2 + 80 vol.% N2 and 70 vol.% CH4 + 30 vol.% CO2 were also studied to mimic industrial applications, namely flue gas and biogas dehydration. The hybrid membranes developed showed barrier properties to all gases studied, with a gas permeability below 1 barrer. On the other hand, they exhibited high water permeabilities and selectivities. When processing the biogas mixture, the water permeability was found to be three times higher than water permeability in the flue gas mixture, leading to a H2O/CH4 selectivity much higher than H2O/N2 selectivity (4042 and 294, respectively). The hybrid FucoPol membranes showed that, in real situations, have the capacity to dehydrate mixtures, with the advantage of not losing N2 or CH4, due to the low permeability values of these gases. This work shows that microbial polysaccharides obtained from a renewable source and purified without using solvents can be a sustainable alternative to other materials used in industrial dehydration processes. Strategies for further improvement should include optimization of polymer cross-linking conditions and optimization of the thickness of the active layer of the composite membranes in order to improve the long term stability of the membranes and promote higher fluxes, without compromising selectivity.

Thauera aminoaromatica MZ1T is an exopolysaccharide (EPS)-producing Gram negative bacterium isolated from the wastewater treatment plant of a major industrial chemical manufacturer as the causal agent for poor sludge dewatering. It shares common features with other known Thauera spp. (i.e. Thauera aromatica, and Thauera selenatis), being capable of degrading aromatic compounds anaerobically and using acetate and succinate as carbon sources. It is unique among the Thauera spp. in its production of abundant EPS which results in viscous bulking and poor sludge dewaterability. In this respect, it is similar to Azoarcus sp. EbN1 and BH72. Thaueran is the proposed name for EPS produced by MZ1T for research purpose. The focus of this research is to fully characterize the microorganism and identify and characterize the genes responsible for thaueran synthesis and export through bioinformatics, transposon mutagenesis, gene clone and expression, reverse transcriptase quantitative real time PCR, and genome sequencing and annotation. Ultimately, this knowledge will contribute to control of viscous bulking and sludge dewatering problems. However, a broader range of important environmental biotechnical processes may be forthcoming from understanding thaueran synthesis. They may include thaueran remedial solutions for heavy metal and radionuclide immobilization, anaerobic carbon channeling and sequestration, greenhouse gas mitigation through acetate incorporation into thaueran, and novel applications such as thaueran-mediated wound healing. Sequencing of MZ1T genome has been accomplished through collaboration with the Joint Genome Institute (JGI). The genome size is 4.5 Mbp, GC content is 68.3%, and there are 4,092 protein coding genes. Three putative thaueran gene clusters were found within the genome. One tight cluster with a size of 20.67kb encoding 14 genes may contain most necessary genes for thaueran formation and export. Another two clusters are loosely organized. Through transposon mutagenesis, mutants not producing abundant thaueran and not flocculating have been obtained and verified, and were further used in reverse transcriptase quantitative real time PCR to compare the differential expression levels of the presumable EPS genes among mutants, wild type MZ1T and under different growth conditions. The results indicated a correlation of the expression level of the test genes and the abundance of EPS.

The overuse of synthetic non-biodegradable plastic packaging in the last decades turned into a serious global environmental problem, once recycling and energy recovery are not perfect solutions. To overcome this problem, efforts have been done by researchers around the world to use biodegradable biopolymers from renewable resources as food packaging materials. In this thesis, FucoPol, a microbial exopolysaccharide has been studied as a sustainable alternative and the following questions were raised: • Is it possible to produce films of FucoPol and which properties should be improved? • Which strategies may be applied to improve their behaviour, specially their barrier properties to gases and water vapour? • Is it possible to use FucoPol films as an alternative to synthetic plastics aiming their use as packaging material? This study shows that, FucoPol films offer good barrier properties to gases and biodegradability. Their weaker properties, low barrier properties to water, have been improved along the work, using two distinct strategies: formulation of bilayer films of FucoPol and chitosan and use of different coatings techniques in FucoPol films. The results have shown that, it is possible to improve FucoPol films properties, namely the barrier properties to gases and water vapour, and resistance to liquid water. FucoPol combined with chitosan as a bilayer film was used for walnuts packaging and it was possible to verify that it can protect food susceptible to lipid oxidation as effectively as synthetic materials.

碩士
國立臺灣海洋大學
海洋生物研究所
102
In this study, effect of UV and diesel oil on the production and composition of exopolysaccharides (EPS) and wax esters by the UV resistant oil degrading bacterium (Ochrobactrum tritice B32), yeast (Candida keelungensis SM22), and two microbial communities (UV12 and UV24) enriched from diesel oil were investigated. Effect of inoculum, medium composition (such as diesel oil concentration, carbon sources or nitrogen sources) and environmental factors (such as temperature, salinity, pH or UV radiation) on the yield and composition of the EPS were determined. Results showed that different carbon sources or nitrogen sources could change the composition (carbohydrate, protein, uronic acid, reducing sugar and lipid) and the emulsifying activity of EPS produced by the microorganisms. These EPSs were found to be completely soluble in distilled water but insoluble in chloroform, toluene and hexane. Maximum EPS production by UV12, UV24, SM22 and B32 were 16.1 mg/mL, 14.0 mg/mL, 15.6 mg/mL and 16.8 mg/mL, respectively.UV (A/B) radiation increased EPS production sharply (1.1-5.2 times) by these microorganisms. UV (A/B) radiation also changed composition of EPS produced by these microorganisms. These EPS were not toxic to Artemia salina nauplii.When testing their ability to remove diesel oil from the contaminated sediment, the results showed that UV radiation could increase marine diesel oil removal. Experimental groups removed 80~95% while the control removed only 71.9% TPH after 70 days inoculation.All the test microorganisms also produced wax esters. In most cases, higher concentration of oil (1-5%) produced higher concentration of wax esters (0.9-4.8 mg/mL). However, when oil concentration increased to 10-20%, wax ester production was not correlated to the oil concentration.The chemical nature and stability of the EPS suggest its potential application in bioremediation of marine environments. Their stability under diverse conditions, such as temperature, pH and salinity, also makes this biopolymer a versatile emulsifier for use in many food and pharmaceutical formulations.

Cheese whey is the main by-product of dairy industries, which is massively produced worldwide. Its disposal may represent a serious environmental issue due to its high organic content, therefore, it is of interest to find more solutions for its valorisation. An interesting approach is the conversion of its lactose into value-added products through microbial fermentation. Kefiran is an exopolysaccharide with approximately equal amounts of D-glucose and Dgalactose, whose remarkable properties make it possible to be of use in a wide range of applications in the medicine, pharmaceutical and food industries. This polymer can be produced during fermentation, with the use of kefir grains, a complex microbial community embedded within a kefiran matrix, containing several species of lactic acid bacteria (LAB), acetic acid bacteria (AAB) and yeasts, living in symbiose. Nevertheless, the bacterium Lactobacillus kefiranofaciens is considered to be the main responsible for kefiran production. The objective of this work was the study of kefiran production, by kefir grains, in cow’s cheese whey (CWC), diluted cow’s cheese whey (CWCD), sheep’s cheese whey (CWS) and in synthetic medium (MRSL broth). In this study, fermentations in batch and fed-batch mode were performed. Initially, MRSL broth medium was used to optimise the process. A fermentation time of 100 h was found to be optimal, since it led to the highest kefiran productivity value. The ideal stirring speed was concluded to be 80 rpm, as it provided the best balance between kefiran production and kefir grains’ conservation. Relatively to the optimal initial lactose concentration, results were dependant on the stirring speed, with no noticeable inhibition by substrate occurring. The best results in terms of kefiran concentration, 1.640 g/L, and productivity, 0.390 g/L.day, were both reached after 100 h of fermentation, using a MRSL broth medium containing 40 g/L of lactose, at 160 rpm. Regarding cheese whey, kefiran production values were promising. The highest concentration of kefiran, 1.190 g/L, was attained in the fed-batch fermentation of the cheese whey sample CWC, which lasted for 510 h, at 80 rpm, while the highest kefiran productivity value, 0.224 g/L.day, was achieved in the batch fermentation of the cheese whey sample CWCD, after 50 h, at 80 rpm. When characterising the kefiran samples produced with the different media, an acid hydrolysis confirmed a glucose to galactose ratio of approximately 1:1 and, together with the analysis by Fourier-transform infrared (FTIR) spectroscopy, also confirmed that kefiran was successfully produced in this study. Additionally, the analysis by scanning electron microscopy (SEM) revealed a foam-like structure with a high porosity level, which is a very desirable characteristic for the production of kefiran scaffolds. This study showed that it is possible to produce kefiran with the use of a lowcost carbon source, such as cheese whey, with fairly solid results, even though the process scale-up still needs to be addressed to enable an eventual industrial implementation.
O soro de leite é o subproduto principal da indústria dos lacticínios, e é massivamente produzido a nível mundial. O seu descarte pode representar um sério problema ambiental devido à sua elevada carga orgânica, portanto, é de interesse procurar mais soluções para a sua valorização. Uma alternativa interessante é a conversão da sua lactose em produtos de valor acrescentado, através da fermentação microbiana. O kefirano é um exopolissacarídeo com aproximadamente as mesmas quantidades de Dglucose e D-galactose, cujas notáveis características tornam possível o seu uso numa ampla gama de aplicações na medicina e na indústria farmacêutica e alimentar. Este polímero pode ser produzido durante a fermentação com grãos de kefir, uma complexa comunidade microbiana embebida numa matriz de kefirano, contendo várias espécies de bactérias do ácido lático (LAB), bactérias do ácido acético (AAB) e leveduras, que vivem em simbiose. Contudo, a bactéria Lactobacillus kefiranofaciens é considerada como sendo a principal responsável pela produção de kefirano. O objetivo deste trabalho foi o estudo da produção de kefirano, utilizando grãos de kefir, em soro de leite de vaca (CWC), soro de leite de vaca diluído (CWCD), soro de leite de ovelha (CWS) e em meio sintético (MRSL broth). Neste estudo, foram realizadas fermentações em modo batch e fed-batch. Inicialmente, o meio MRSL foi utilizado para a otimização do processo. O tempo de fermentação de 100 h foi considerado como ótimo, porque resultou no valor de produtividade de kefirano mais elevado. A agitação ideal foi de 80 rpm, uma vez que demonstrou o melhor balanço entre a produção de kefirano e a conservação dos grãos de kefir. Relativamente à ótima concentração inicial de lactose, os resultados foram dependentes da agitação, onde não foi observada uma inibição por substrato significativa. Os melhores resultados em termos de concentração de kefirano, 1,640 g/L, e produtividade, 0,390 g/L.dia, foram ambos alcançados após 100 h de fermentação, usando um meio MRSL broth contendo 40 g/L de lactose, a 160 rpm. Em relação aos ensaios com soro de leite, os valores de produção de kefirano foram promissores. A concentração mais alta de kefirano, 1,190 g/L, foi atingida na fermentação em fed-batch da amostra de soro de leite CWC, que durou 510 h, a 80 rpm, enquanto que o valor mais alto de produtividade de kefirano, 0,224 g/L.dia, foi alcançado na fermentação em batch da amostra de soro de leite CWCD, depois de 50 h, a 80 rpm. Ao caracterizar as amostras de kefirano produzidas com os diferentes meios, uma hidrólise ácida confirmou o rácio de glucose para galactose de aproximadamente 1:1 e, juntamente com a análise por espectroscopia de infravermelhos com transformadas de Fourier (FTIR), também confirmou que o kefirano foi produzido com sucesso neste estudo. Adicionalmente, a análise por microscopia eletrónica de varrimento (SEM) revelou uma estrutura tipo espuma com um elevado nível de porosidade, o que constitui uma característica muito desejável para a produção de scaffolds de kefirano. Este estudo demonstrou que é possível produzir kefirano a partir de fontes de carbono de baixo custo, como o soro de leite, com resultados bastante sólidos, apesar de ainda ser necessário estudar o aumento de escala do processo, de modo a tornar possível uma eventual implementação industrial
Mestrado em Biotecnologia