Dissertations / Theses on the topic 'Lactate permease'

Non-typeable Haemophilus influenzae (NTHi) is a small acapsulate gram-negative coccobacillus and a human restricted nasopharynx commensal. Nevertheless, it is the main cause of chronic obstructive pulmonary disease (COPD) exacerbation. A previous dual RNA-sequencing study highlighted the upregulation of an L-lactate permease gene, suggesting a role for environmental lactate during the infection. Furthermore, a strong downregulation of the sialic acid-specific tripartite ATP-independent periplasmic transporter (TRAP system) was reported, while a gene (pst) with 60% similarity with nanT, an N-acetyl neuraminic acid (NANA) transporter in E. coli, was found to be upregulated during the infection. In order to explore the impact of environmental lactate and NANA utilization during NTHi mucosal colonization, we constructed specific knock out strains for two uptake systems and analysed bacterial fitness and their resistance to complement-mediated killing during infection of human bronchial epithelia in vitro. In vitro assays were performed to analyse NTHi Δpst resistance to serum killing. The pst gene mutant strain did not show any alteration in the complement mediated killing resistance, even when the test was performed after an adaptation of the bacterium by infection of human bronchial epithelial cells. These data suggested a lack of mutuality between the TRAP system and the pst gene as sialic acid transporter during the infection stages. For what concerns the lactate permease knock out (ΔlctP) we observed a faster grow rate compared to the WT strain when lactate is the sole available carbon source. We also reported an impaired membrane composition which may cause this cell instability, more precisely we showed a lipid A decrement in a “lactate environment”. The altered membrane composition results in a strong aggregative phenotype both in bacteria-bacteria and bacteria-epithelia interactions. Despite the low endotoxin levels NTHi ΔlctP was still able to resist to the complement mediated killing when NANA was available. These data open to unknown complement killing evasion systems. In conclusion, our work is aimed to better characterize NTHi infection factors involved in the colonization of the human epithelium.

Valorizacija permeata kao sporednog proizvoda industrije mleka je od izuzetnog ekološkog, ekonomskog i tehnološkog značaja.Cilj doktorske disertacije je razvoj tehnološkog procesa prerade permeata, kao sporednog proizvoda dobijenog nakon ultrafiltracije mleka tokom proizvodnje feta sira i svežeg („mladog“) sira. Ispitana je mogućnost enzimske hidrolize laktoze u permeatu korišćenjem enzima β-galaktozidaze izolovanog iz Kluyveromyces lactis u koncentraciji 0,1, 0,3 i 0,5 g/100g na temperaturama 20º, 30º i 40 ºC. Praćene su promene sadržaja laktoze, D–galaktoze i D–glukoze u vremenskim intervalima tokom 60 minuta. Posebna faza istraživanja obuhvatila je matematičko modelovanje i kinetiku procesa hidrolize laktoze u permeatu pod dejstvom β –galaktozidaze i primenu hidrolizovanog permeata u proizvodnji mlečnih napitaka po odabranoj formulaciji. Predložen je tehnološki proces proizvodnje napitka na bazi hidrolizovanog permeata sa dodatkom voćnih baza. Utvrđeni su parametri kvaliteta i trajnosti napitaka tokom 60 dana skladištenja.Na temperaturi 40°C dodatkom enzima β -galaktozidaze u koncentraciji 0,1g/100g za 60 minuta postiže se 100% stepen hidrolize prisutne laktoze u permeatu. Sa većom koncentracijom enzima, 0,3 g/100g odnosno 0,5g/100g, na istoj temperaturi, isti efekat se postiže za 20 minuta.Ispitivanjem kinetike hidrolize laktoze potvrđena je kinetika prvog reda. Generalno posmatrano visoki koeficijenti determinacije pokazuju dobro poklapanje eksperimentalnih rezultata i matematičkog modela reakcije prvog reda. Vrednosti se kreću od 0,974 (temperatura 20°C) do preko 0,990 (na temperaturama 30°C i 40°C) pri koncentraciji enzima 0,1g/100g.Proizvedeni napici od hidrolizovanog permeata su delaktozirani i ne sadrže mlečnu mast. Od ukupnih šećera u svim napicima više od 50% čini glukoza: 50,16% - napitak šumsko voće, 50,42% - napitak pomorandža/šargarepa, 54,65% - napitak multivitamin, odnosno 55,13% - napitak crveno voće.Najveći sadržaj vitamina C nakon proizvodnje imao je napitak sa dodatkom voćne baze multivitamin 0,3972 mg/100g, zatim šumsko voće 0,2887 mg/100g i pomo-randža/šargarepa 0,1999 mg/100g.Najveću vrednost antioksidativne aktivnosti nakon proizvodnje pokazali su uzorci napitka sa multivitaminom i šumskim voćem. Tokom perioda skladištenja dolazi do smanjenja DPPH vrednosti. Najmanji pad je u napitku sa pomorandžom / šargarepom (smanjenje za 17%), a najveći u napitku sa šumskim voćem (za 39%). Analizirani uzorci sadrže ukupnih polifenola u intervalu od 47,84 do 120,38 mg GAE/l u zavisnosti od vrste napitka, odnosno dodatih voćnih baza.Generalno može se zaključiti da se prime-njenim tehnološkim procesom dobijaju napici stabilnog fizičko-hemijskog sastava tokom 60 dana skladištenja, visoke nutritivne i niske energetske vrednosti.
Valuation of the permeate as a by-product of the dairy industry is of great ecological, economic and technological importance.The aim of the PhD thesis is the development of the technological process of refining permeate, as a by-product obtained after ultrafiltration of milk during the production of feta cheese and fresh cheese. The possibility of enzymatic hydrolysis of the lactose in the permeate using the enzyme β-galactosidase isolated from Kluyveromyces lactis in a concentration of 0.1, 0.3 and 0.5 g / 100 g at a temperature of 20°, 30° and 40° C was examined. Changes in the content of lactose, D-galactose and D-glucose at intervals of 60 minutes were monitored. A special stage of the research included mathematical modeling and kinetics of lactose hydrolysis in the permeate under the influence of β-galactosidase and application of hydrolyzed permeate in the production of dairy products under the selected formulation. A technological process of producing a beverage on the basis of hydrolyzed permeate with the addition of fruit bases was suggested. Quality and durability parameters were determined for drinks during the 60 days of storage.Addition of the enzyme β-galactosidase at a concentration of 0.1 g / 100 g for 60 minutes at a temperature of 40 ° C a 100% degree of hydrolysis of lactose is achieved, present in the permeate. With a higher concentration of enzyme, 0.3 g / 100 g or 0.5 g / 100g, at the same temperature, the same effect can be achieved in 20 minutes.By examining the kinetics of lactose hydrolysis the first order kinetics was confirmed. Generally high coefficients of determination show good correspondence between the experimental results and the mathematical model of the first order reaction. Values range from 0.974 (at a temperature of 20° C) up to over 0.990 (at temperatures 30° C and 40° C) at a an enzyme concentration of 0.1g / 100g.Beverages produced from hydrolyzed permeate are lactose-free and fat-free products. More than half of the total sugar content in all beverages consists of glucose: 50.16%-forest fruit beverage, 50.42%-beverage orange/carrot, 54.65% beverage multivitamin and 55.13% - beverage red fruit.The highest vitamin C content after production was in a beverage with the addition of fruit base multivitamin (0.3972 mg/100g), followed by forest fruit (0.2887 mg/100g) and orange/carrot (0.1999 mg/100g).Beverage samples with multivitamin and forest fruits showed the highest value of antioxidant activity after production. During the storage period there is a reduction of DPPH values. The smallest decrease was in the beverage with orange/carrot (decreased 17%), and the biggest in the beverage with forest fruit (39%). The content of polyphenols in analyzed samples ranges from 47.84 to 120.38 mg GAE/L depending on the type of beverage and added fruit base.Overall it can be concluded that the applied technological process gives beverages of stable physical and chemical content during the 60 days of storage, of high nutritional value and low energy.

Orientador: Salvador M. Roig
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
Made available in DSpace on 2018-07-20T23:21:40Z (GMT). No. of bitstreams: 1 Costa_RicardoCalvo_M.pdf: 2710464 bytes, checksum: 2f1749d07b2a3e39917bedff151f819f (MD5) Previous issue date: 1995
Resumo: Neste trabalho foi estudada a extração de lactose a partir de permeado de leite e de permeado de soro de queijo, obtidos por ultrafiltração de leite e soro de queijo. A antecipação da etapa de descoloração foi estudada com o objetivo de eliminar a etapa de refino no processo tradicional e obter uma lactose de alto teor de pureza. A pasta descorante composta de 750/0 de carvão ativo e negro de ossos e 25% de ácido clorídrico concentrado foi adicionada aos permeados de leite e de soro de queijo, e somente resultou em descoloração quando foram utilizados teores de pasta superiores a 8% da massa de lactose presente no permeado. Os melhores resultados foram obtidos a partir de permeado de leite, obtido através de ultrafiltração de leite em um sistema de ultrafiltração dotado de membranas minerais, sem descoloracão do permeado. Os compostos coloridos foram retidos durante o processo de ultrafiltração resultando em um permeado límpido. O melhor processo obtido para extração de lactose a partir de permeado foi ultrafiltração do leite em membrana mineral, seguido de concentração à vácuo do permeado, cristalização, separação, lavagem dos cristais com água a 5°e e secagem. A partir de permeado de leite com 0,027% de nitrogênio total 0,49% de cinzas e 4,71 % de lactose foi obtida lactose com 99,3% de pureza 0,66% de cinzas e 0,07% de nitrogênio total
Abstract: The lactose extraction by ultrafiltration from milk and whey permeates was studied. The aim of this work was to eliminate the refining step of the traditional process and produce a high purity level lactose yield before the discolouring step. The discolouring paste which is made of 75% of a mixture of active carbon and black bone and 25% of concentrated hydrogen chloride was added to milk and whey permeates resulting in discolouring only when the paste concentration was higher than 8% in relation to the lactose mass in the permeate. The best results carne from milk permeate processed in a mineral membranes milk ultrafiltration system without the discolouring step. The colouring compounds were retained in the ultrafiltration process resulting in a cleaned permeate. The best process to lactose extraction from permeate was milk ultrafiltration in a mineral membrane system followed by permeate vacuum concentration, crystallization, separation and crystal washing with 't\1Iter at 5°e and drying. From mill permeate with 0,027% total nitrogen, 0,49% ash and 4,71% of lactose it was possible to obtain lactose with a purity of 99,3%, 0.660% ash and 0,07% total nitrogen contents
Mestrado
Mestre em Tecnologia de Alimentos

The interaction between a membrane protein and its surrounding phospholipids is thought to be crucial for the correct folding and function of the protein. This thesis is focused on the investigation of the interplay between Lactose permease (LacY), a paradigm for secondary transporters present in the inner membrane of Escherichia coli and model systems mimicking its natural lipid environment. Since the role of phospholipids in LacY’s activity is currently being refined, this work represents a contribution to the field by studying the interaction at two different levels: (i) the LacY interplay with the phospholipids present at the annular region in the vicinity of the protein was studied through FRET measurements between a single-tryptophan LacY mutant and diverse pyrene-marked phospholipids, and (ii) the LacY interaction with the more distanced bulk phospholipids was studied through supported proteo-lipid sheets that were analysed using topography, force-spectroscopy and force-volume Atomic Force Microscopy modes. First, after validating LacY preference for phospholipid fluid (Lα) phases in the studied two-component model systems, a different composition between bulk and annular regions was confirmed. Hence, bulk lipids, which were assimilated to the phospholipids in Lα phase, were mainly formed by PG, while PE was the main component of the annular region. This points to a direct annular phospholipid-LacY selectivity because it discards a random phospholipid distribution near the protein. Second, the LacY selectivity for precise phospholipid species at the annular region was found to be related to: (i) a neutral charged phosholipid (PE or PC, with preference for the former), and (ii) phospholipids with large negative spontaneous curvature (C0) (DOPE > POPE). In addition, D68 was revealed as an important amino acid for the protein annular lipid selectivity. Third, the interaction between LacY and the bulk lipids was described as reciprocal. Accordingly, the presence of the protein largely modified the topography and the nanomechanics of the lipid system, especially for the Lα phase, whilst the nanomechanics of LacY itself were different depending on the surrounding lipid matrix: more force was needed to pull LacY form the DPPE:POPG (3:1, mol/mol) system than from the POPE:POPG (3:1, mol/mol) one. Therefore, the bilayer lipid composition seems to determine the forces governing the LacY tight interaction with the membrane and can be thus decisive for the protein correct insertion and activity.
La interacció entre una proteïna de membrana i els fosfolípids que l’envolten és crucial pel bon plegament i la correcta funció de la proteïna. Aquesta tesi està centrada en la investigació de la interacció entre la Lactosa permeasa (LacY), un paradigma dels transportadors secundaris situat a la membrana interna d’Escherichia coli, i sistemes models que mimetitzen el seu entorn lipídic. Aquest treball representa una contribució al camp a través de l’estudi de la interacció a dos nivells: (i) la interacció entre LacY i els fosfolípids presents a la regió anular propera a la proteïna ha estat estudiada a través de mesures de FRET entre un mutant de LacY amb un únic triptòfan i diversos fosfolípids marcats i (ii) la interacció entre LacY amb els fosfolípids més llunyans o bulk s’ha investigat a través de làmines de lípid i proteïna sobre un suport, les quals s’han analitzat a partir de diversos modes de microscòpia de força atòmica (topografia, espectroscòpia de força i force-volume). En primer lloc, s’ha validat la preferència de LacY pels fosfolípids en fases fluïdes (Lα). A més, s’ha confirmat una composició lipídica entre la regió anulars i el bulk. Així, els fosfolípids bulk, considerats com a fosfolípids en fase Lα, tenen PG com a principal component, mentre que PE és el major component de la regió anular. Això sembla indicar una selectivitat entre LacY i els fosfolípids anulars. En segon lloc, s’ha descrit que la selectivitat de LacY per fosfolípid determinat a la regió anular està relacionada amb (i) càrrega neutra i (ii) curvatura espontània (C0) negativa. A més, D68 s’ha assenyalat com un aminoàcid important per la selectivitat de la proteïna envers els lípids anulars. Finalment, s’ha descrit una interacció recíproca entre LacY i els lípids bulk. Així, la presencia de la proteïna modifica la topografia i la nanomecànica del sistema lipídic, especialment de la fase Lα, i, alhora, la nanomecànica de la pròpia LacY varia segons la matriu lipídica que l’envolta. En conseqüència, la composició lipídica de la bicapa sembla determinar les forces que governen l’estreta interacció de LacY amb la membrana i, per tant, aquesta composició és decisiva per la correcta inserció i activitat de la proteïna.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2015-11-09T13:45:39Z No. of bitstreams: 1 texto completo.pdf: 600665 bytes, checksum: ee26156d6dc7124f99efde31caf1f198 (MD5)
Made available in DSpace on 2015-11-09T13:45:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 600665 bytes, checksum: ee26156d6dc7124f99efde31caf1f198 (MD5) Previous issue date: 2010-04-12
Fundação de Amparo à Pesquisa do Estado de Minas Gerais
A permease de lactose de Kluyveromyces lactis, Lac12, media o transporte de lactose e o de galactose de baixa afinidade. Aqui é apresentado o estudo do efeito de fontes de carbono na internalização de Lac12 através do uso de linhagens contendo o gene quimérico LAC12-GFP. Quando células de K. lactis pré-cultivadas em galactose ou lactose foram transferidas para um novo meio, Lac12-GFP foi removida da membrana plasmática e localizada intracelularmente. Surpreendentemente, mesmo a presença de galactose ou lactose no novo meio de transferência causou essa internalização, e a resposta celular foi diferente para esse dois açúcares. Os resultados obtidos revelam que o processo de internalização é dependente do tipo de açúcar presente e de sua concentração. A internalização de Lac12-GFP causou redução nas taxas de captação de lactose[C14] e também foi observada em uma linhagem mutante Klsnf1; portanto, esse evento independe da atividade de KlSnf1. Evidências indicam que glicose-6-fosfato é o sinal intracelular, uma vez que a internalização foi induzida por 2-deoxiglicose, e a inibição da atividade da enzima fosfoglimutase por lítio impediu a internalização por galactose, mas não por lactose ou glicose. A internalização não ocorreu em 6-deoxiglicose, e, em ausência de síntese protéica, o evento foi irreversível.
Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we have investigated the effects of carbon sources on internalization of Lac12 by using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and localized intracellularly. Surprisingly, even galactose or lactose in the new media caused the internalization, and cells responded differently to theses two sugars. Our results reveal that this process is dependent of sugar species and also sugar concentration. Lac12-GFP internalization causes reduction on [C14]lactose uptake rates and also occurs in a Klsnf1 mutant strain; thereby, it is independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, since internalization was induced by 2-deoxyglucose and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in absence of protein synthesis.

This study aimed to obtain 15% total solids and reduced mineral content in milk UF permeate by nanofiltration, hydrolysing the lactose content of nano-concentrate enzymically, partially substituting sucrose in ice cream formulations with hydrolysed lactose nano-concentrate (HLNC), and investigating the effects of HLNC on the physio-chemical and sensory characteristics of ice cream. The desired 15% total solids in the nano-concentrate was achieved after three fold concentration of milk UF permeate. The colour of milk permeate changed, pH and mineral content decreased, and crude protein content, lactose content and titratable acidity increased. The lactose content was hydrolysed by enzyme lactase. HLNC was used to replace 25% and 50% of sucrose in ice cream formulations. Springiness, cohesiveness, chewiness, adhesiveness, hardness, iciness, Ph and colour were not significantly affected. Viscosity, freezing point, glass transition temperature, melting temperature, gumminess and sweetness were significantly decreased, whereas freezing time, saltiness and cooked flavour were significantly increased. The overall acceptability of ice cream significantly decreased at 50% but was insignificantly affected at the 25% level. These results indicate that about one quarter of sucrose could be replaced by HLNC.
Master of Science (Hons) (Food Technology)

Proteins are essential in all living organisms. They are involved in various critical activities and are also structural components of cells and tissues. Lactose permease a membrane protein has become a prototype for the major facilitator super family and utilises an existing electrochemical proton gradient to shuttle galactoside sugars to the cell. Therefore it exists in two principle states exposing the internal binding site to either side of the membrane. From previous studies it has been suggested that protonation precedes substrate binding but it is still unclear why this has to occur in the event of substrate binding. Therefore this study aimed to bridge this gap and to determine the chemical characteristics of the transport pathway. Molecular dynamics simulation methods and specialised simulation hardware were employed to elucidate the dependency of substrate binding on the protonation nature of Lactose permease. Protein models that differed in their conformation as well as their protonation states were defined from their respective X-ray structures. Targeted molecular dynamics was implemented to drive the substrate to the binding site and umbrella sampling was used to define the free energy of the transport pathway. It was therefore suggested that protonation for sugar binding is due to the switch-like mechanism of Glu325 in the residue-residue interaction (His322 and Glu269) that leads to sugar binding only in the protonated state of LacY. Furthermore, the free energy profile of sugar transport path way was lower only in the protonated state which indicates stability of sugar binding in the protonated state.

Orientadores: Valdemiro Carlos Sgarbieri, Alda Luiza Santos Lerayer
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
Made available in DSpace on 2018-08-02T22:36:22Z (GMT). No. of bitstreams: 1 Saron_MargarethLopesGalvao_M.pdf: 23626715 bytes, checksum: 97334ba53a2521a3bbac09b596a00d1f (MD5) Previous issue date: 2003
Resumo: O presente trabalho teve por objetivo estudar novas aplicações para o permeado proveniente da concentração das proteínas do soro de leite bovino pelo processo de ultrafiltração em membrana (PM de corte de 10 Kda). A concentração do permeado, evaporação sob vácuo permitiu a insolubilização da lactose e sua purificação. Através de reação de isomerização em pH alcalino e o uso de borato de sódio como catalizador, obteve-se a conversão da lactose em lactulose e sua purificação por cromatografia de troca iônica e exclusão molecular em colunas. A lactulose purificada foi testada in vitro como prebiótico em vários meios de cultura complementados com o permeado de soro de leite, para o crescimento das bactérias probióticas (Lactobacillus acidophilus La-5 e Bifidobacterium /actis Bb-12). Verificou-se que tanto o permeado de soro de leite quanto a lactulose funcionaram como importantes fatores de crescimento para essas bactérias. O permeado de soro contem aproximadamente 85% de lactose (em base seca), sendo bastante deficiente em aminoácidos. Após separação da lactose, o líquido sobrenadante do permeado representa uma boa fonte de vitaminas, particularmente B1, B2 e niacina. A cromatografia em camada delgada (CCO) foi eficiente para a detecção da lactulose após eluição em colunas e, através da cromatografia líquida de alta eficiência (CLAE) pode-se confirmar a reação de isomerização da lactose em lactulose com rendimento de aproximadamente 70%. Os ensaios microbiológicos mostraram que a complementação do meio MRS (De Man, Rogosa e Sharpe) com permeado de soro de leite (PSL) promoveu um aumento significativo na produção de células de L.acidophi/us e B./actis. O PSL, quando combinado com extrato de levedura (EXT) na proporção de 75/25, mostrou-se adequado para a propagação de células de B. /actis. O meio MRS adicionado de 2% ou 3% de lactulose apresentou um aumento do crescimento de L. acidophi/us. A concentração de apenas 1% de factulose no meio MRS modificado e PSUEXT foi eficiente para estimular o rescimento de Bifidobacterium /actis. Tanto o L. acidophilus como a B. /actis demonstraram resistência as condições de acidez (pH 2 , 3h, 37°C). A presença de lactulose no meio de cultura promoveu maior proteção destas bactérias quando expostas à presença de sais biliares. Neste trabalho são descritos ainda aspectos relevantes dos efeitos benéficos dos prebióticos e probióticos para a saúde humana.
Abstract: The objetive of the present work was to study new application for the permeate obtained during concentration of bovine milk whey by the process of membrane ultrafiltration (MW cut off 10 Kda). The concentration of the permeate, by vacuum evaporation, permited the insolubilization and further purification of the lactose by crystallization. By an isomerization reaction at alkaline pH using sodium borate as catalyst, the conversion of lactose to lactulose was promoted which was purified in ionic exchange and molecular exclusion colunn chromatography. The purified lactulose was tested in vivo as prebiotic in various culture media complemented whith whole whey permeate, for the growth of the probiotic bacteria Lactobacillus acidophilus La-5 and Bifidobacterium lactis Bb-12. It was verified that both the whole permeate as well as the lactulose functioned as important growth promoting factors for these bacteria. The whey permeate contained approximately 85% lactose on a dry basis and was deficient in amino acids. After lactose separation the permeate supernatant liquid contained appreciable quantities of vitamins, particularly B1, B2 and niacin. Thin layer chromatography (TLC) was efficient in the detection of lactulose after elution from the columns, and. high perfomance liquid chromatography (HPLC) was used to confirm the isomerization reaction of lactose to lactulose whith a yield of approximately 70%. The microbiological assay showed that complementation of the MRS (De Man, Rogosa and Sharpe) medium whith whole milk whey permeate significant increase in cell production of both L. acidophilus and B. lactis. The modified MRS medium (MRSm) added of 2% or 3% lactulose showed an increase in growth for the L. acidophilus. At only 1% lactulose concentration in the MRSm or MWPNE media a significant increase in growth was observed for B. lactis. Both L. acidophilus and B. lactis showed resistance to acidity condition (pH 2, 3h, 37°C). The presence of lactulose in the culture media promoted higher protection to these bacteria when exposed to biliary salts. In this work important aspects of the beneficial effects of prebiotics and probiotics to human health were described.
Mestrado
Mestre em Alimentos e Nutrição

The current energy crisis is exponentially growing and widening the chasm between demand and supply. Biofuels such as ethanol not only provide greener alternatives to fossil fuels but have been shown to reduce emissions from vehicles, improving air etc. Biofuel production from sources such as cellulose is believed to be more sustainable due to its low cost, vast availability in nature and sources such as industrial plant waste can be put to good use. However, due to the absence of a low-cost technology to overcome its recalcitrance, a concept called Consolidated Bioprocessing (CBP) has been put forward which proposes to integrate the production of saccharolytic enzymes, hydrolysis of the carbohydrate components to sugar molecules, and the fermentation of hexose and pentose sugars to biofuels into a single process. The present study involves development of cellulolytic E. coli strains towards cellodextrin assimilation by employing an energy-saving strategy in cellulose metabolism through the phosphorolytic cleavage of cellodextrin mixture produced as cellulosic degradation products.

Dissertação composta por 2 artigos.
Capes
5000
O soro lácteo é produzido pela indústria de laticínios durante a fabricação de queijos. Contém aproximadamente 80% da lactose original além de minerais, vitaminas, aminoácidos livres, peptídeos e outros compostos de baixo peso molecular. Porém, muitas indústrias ainda consideram o soro como um efluente, o qual quando não devidamente tratado gera um sério problema ambiental por causa de sua elevada carga orgânica. Estes fatores tornam importante o desenvolvimento de alternativas para um adequado aproveitamento do soro, porque ao mesmo tempo em que a transformação do soro em produtos diversos diminui o problema ambiental, proporciona ganhos às indústrias de laticínios, através do desenvolvimento de novos produtos. Uma técnica bastante vantajosa para reaproveitamento do soro é o processo de ultrafiltração, para a obtenção do concentrado proteico do soro do leite, um produto bastante valorizado na indústria alimentícia. Este processo gera outro coproduto, o permeado do soro do leite, que é rico em lactose, a qual pode ser convertida em lactulose, um prebiótico, capaz de ser metabolizado no intestino grosso por bactérias probióticas. Este trabalho teve como objetivo principal otimizar um método de síntese para a produção de lactulose a partir do permeado do soro de leite bovino. A metodologia de superfície de resposta foi utilizada para investigar o efeito de dois parâmetros (tempo e isomerizante) na isomerização da lactose do permeado de soro para a produção lactulose. A experimentação teve como objetivo definir as faixas ótimas de operação para as variáveis do processo, visando o melhor rendimento da lactose. Os resultados demonstraram que o fator isomerizante foi o mais significativo, e a utilização do ácido bórico para este fim contribuiu positivamente para aumentar o rendimento, enquanto que o efeito tempo não foi significativo, mas indicou que um aumento no tempo de reação influencia negativamente na resposta. A partir da pesquisa, verificou-se ser possível o processo de isomerização do permeado de soro de leite para a obtenção da lactulose, a qual tem uma grande aplicação na indústria alimentícia e farmacêutica, sendo também uma alternativa viável para a problemática do meio ambiente.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T20:05:11Z No. of bitstreams: 1 texto completo.pdf: 217487 bytes, checksum: 5604261524efd57a87ca9eed998aafa3 (MD5)
Made available in DSpace on 2017-06-13T20:05:11Z (GMT). No. of bitstreams: 1 texto completo.pdf: 217487 bytes, checksum: 5604261524efd57a87ca9eed998aafa3 (MD5) Previous issue date: 2004-02-16
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Com o objetivo de estabelecer a condição fisiológica para a conversão máxima da lactose do permeado de soro de queijo em etanol, avaliaram-se os efeitos da concentração de substrato e do nível de oxigênio no direcionamento do fluxo metabólico para a via fermentativa da levedura K. marxianus UFV-3. Inicialmente, as fermentações foram conduzidas em meio YNB sintético com altas concentrações de lactose, em condições aeróbica e microaeróbica (injeção de nitrogênio gasoso por 12 minutos), em regime de batelada. Nas fermentações sob microaerobiose, os rendimentos máximos de etanol por substrato foram próximos a 85% do rendimento teórico, ou seja, superiores aos obtidos em aerobiose, por volta de 55% desse rendimento. O rendimento de etanol máximo por massa celular também foi maior em microaerobiose, sendo a produtividade volumétrica maior em microaerobiose apenas na fermentação, cuja concentração inicial de lactose foi de 67,0 g L -1 . As fermentações realizadas em permeado de soro de queijo com 10 diferentes concentrações de lactose, que variaram de 1,0 g L -1 a 240,0 g L -1 , em regime de batelada, sob condições aeróbica, microaeróbica e anaeróbica (injeção de nitrogênio gasoso por todo o tempo de fermentação), apresentaram rendimento máximo de etanol por substrato e por massa celular, bem como de produtividade máxima volumétrica, superior àqueles obtidos em YNB. Além disso, K. marxianus UFV- 3 exibiu maiores velocidades específicas de crescimento, maior produção de massa celular e maior produção de glicerol nas fermentações em permeado que naquelas em YNB. Os parâmetros fermentativos da produção de etanol em permeado foram maiores em microaerobiose e anaerobiose do que em aerobiose. As fermentações em permeado – cujas concentrações iniciais de lactose foram acima de 50 g L -1 – e o nível de oxigênio reduzido (microaerobiose e anaerobiose) apresentaram altos rendimentos máximos de etanol por substrato, correspondendo a quase 100% do teórico. A velocidade de consumo de lactose e de produção de etanol aumentou à medida que a concentração inicial de substrato também aumentou e o nível de oxigênio diminuiu. A tolerância do produto (etanol) foi verificada no permeado acrescido de etanol, em concentrações que variaram de 5 a 80 g L -1 . Em 50 gL -1 , K. marxianus UFV-3 manteve um crescimento próximo a 80% em relação ao controle em condição anaeróbica e perto de 60% sob condições microaeróbica e aeróbioca.
To establish the physiological condition for maximum conversion of lactose from permeated of cheese whey in ethanol, the effects of substrate concentration and level of oxygen in the directioning of the metabolic flow for the fermentative pathway of the yeast K. marxianus UFV-3 were evaluated. Initially, fermentation was carried out in synthetic YNB medium with high lactose concentrations, in aerobic and microaerobic conditions (injection of nitrogen gas for 12 minutes), in batch culture. For fermentation under microaerobiosis, the maximum ethanol yield per substrate was close to 85% of the theoretical yield, in other words, superior to the obtained in aerobiosis, about 55% of that yield. The maximum ethanol yield per cellular mass was also higher in microaerobiosis, being the volumetric yield higher in microaerobiosis only in fermentation, with initial lactose concentration of 67,0 g L -1 . The fermentation performed in permeated of cheese whey with 10 different lactose concentrations which that varied from 1,0 g L -1 to 240,0 g L -1 , in batch regime, under aerobic, microaerobic and anaerobic conditions, (injection of nitrogen gas xifor the entire fermentation) presented maximum ethanol yield per substrate and per cellular mass, as well as maximum volumetric yield, higher than those obtained in YNB. Besides, K. marxianus UFV-3 gave greater specific growth rates, greater cellular mass production and greater glycerol production in fermentation in permeated than in YNB. The fermentative parameters of ethanol production in permeated were greater in microaerobiosis and anaerobiosis than in aerobiosis. The fermentation in permeated – with initial lactose concentrations above 50 g L -1 – and reduced oxygen level (microaerobiosis and anaerobiosis) gave high ethanol maximum yield per substrate, corresponding to almost 100% of the theoretical value. The lactose consumption rate and ethanol production rata increased with the increase in the initial substrate concentration and the oxygen level decreased. The tolerance of the product (ethanol) was verified in permeated added to ethanol, in concentrations ranging from 5 to 80 g L -1 . In 50 gL -1 , K. marxianus UFV-3 maintained a growth close to 80% in relation to the control in anaerobic conditions and close to 60% under microaerobic and aerobic conditions.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2014-09-22T17:50:15Z No. of bitstreams: 3 license_text: 22228 bytes, checksum: 65eccae6ba48e4ed6b2a75cb6a5f37bb (MD5) license_rdf: 21686 bytes, checksum: f60c8e7b7ea9f3ba141b21b00747aece (MD5) 2014ElvioLeandroBurlani.pdf: 2196003 bytes, checksum: d505b809756057eda4fc0e9260181cf5 (MD5)
Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2014-09-29T13:14:40Z (GMT) No. of bitstreams: 3 license_text: 22228 bytes, checksum: 65eccae6ba48e4ed6b2a75cb6a5f37bb (MD5) license_rdf: 21686 bytes, checksum: f60c8e7b7ea9f3ba141b21b00747aece (MD5) 2014ElvioLeandroBurlani.pdf: 2196003 bytes, checksum: d505b809756057eda4fc0e9260181cf5 (MD5)
Made available in DSpace on 2014-09-29T13:14:40Z (GMT). No. of bitstreams: 3 license_text: 22228 bytes, checksum: 65eccae6ba48e4ed6b2a75cb6a5f37bb (MD5) license_rdf: 21686 bytes, checksum: f60c8e7b7ea9f3ba141b21b00747aece (MD5) 2014ElvioLeandroBurlani.pdf: 2196003 bytes, checksum: d505b809756057eda4fc0e9260181cf5 (MD5)
No setor industrial muitos resíduos gerados são tratados e posteriormente descartados nos cursos hídricos. Na indústria láctea para produção de um quilo de queijo são gerados nove litros de soro de queijo, resíduo de elevada carga orgânica, rico em aminoácidos essenciais e vitaminas de importância nutricional. Algumas formas de aproveitamento do soro de queijo são a produção de ricota e de concentrado proteico de soro. Porém esses processos geram outros dois resíduos, respectivamente, o soro de ricota e o permeado de soro de queijo, que são importantes contaminantes ambientais devido à sua elevada carga orgânica. O principal constituinte desses resíduos é a lactose, açúcar que pode ser transformado através de processos fermentativos com auxílio de leveduras, em etanol. Este trabalho teve como objetivo utilizar o soro de ricota e o permeado de soro de queijo para a produção de bioetanol, através do emprego de diferentes cepas da levedura Kluyveromyces spp. Inicialmente foi selecionada entre cinco cepas de leveduras, quatro Kluyveromyces marxianus e uma Kluyveromyces lactis, a que apresentava maior produção de etanol a partir do soro de ricota e permeado de soro de queijo. Nessa etapa foi avaliado também o emprego dos subprodutos, soro de ricota e permeado de soro, nas formas autoclavado e não autoclavado. Posteriormente, empregando a cepa selecionada e a metodologia de planejamento experimental, foram estudados os efeitos do pH inicial, temperatura de incubação e concentração inicial de lactose sobre a produção de etanol, tanto para o soro de ricota e permeado de soro de queijo. Após, avaliou-se em biorreator de 3 L a conversão da lactose em etanol pela cepa selecionada para ambos os subprodutos. Como última etapa do trabalho realizou-se a estimativa de investimento em uma estação de tratamento de efluentes (ETE) e em uma usina de biotransformação de soro de ricota e permeado de soro de queijo. A melhor produção de etanol foi com soro de ricota e permeado de soro de queijo autoclavados utilizando a cepa da levedura Kluyveromyces marxianus ATCC 46537, que produziu 15,75 e 10,40 g/L de etanol, respectivamente. No planejamento experimental foi observada que a fermentação da lactose presente no soro de ricota e permeado de soro de queijo foi mais eficiente com temperaturas entre 35 e 45º C e pHs entre 4 e 5. Com esse estudo foi possível estimar que o investimento de uma usina pode ser viável ao longo de 10 anos, mesmo com um custo elevado de investimento. Além disso, a usina gera lucro, já na ETE o investimento para a instalação é alto e não gera lucro. Os resultados obtidos indicam que é possível obter etanol a partir da lactose presente no soro de ricota e no permeado de soro de queijo empregando a levedura Kluyveromyces marxianus ATCC 46537.

Cette thèse concerne le développement d’approches spectroscopiques infrarouge et Raman exaltées de surface: la spectroscopie infrarouge exaltée de surface (SEIRAS) combinée avec une cellule de perfusion et la spectroscopie Raman exaltée de surface (SERS) couplée avec l’électrochimie. Dans le cadre du premier projet, différentes protéines ont été étudiées : lactose perméase (LacY), complexe I et IM30. Nous avons déterminé le pKa de Glu325 dans LacY sauvage et dans différents mutants portant des mutations dans le centre actif de translocation des protons. Sauvage complexe I a été oxydé avec différents agents oxydants et réduit avec NADH. Spectres différentiels correspondants ont été analysés. Des changements conformationnels dans la protéine IM30, induits par la présence des ions Mg2+, ont été observés.Dans le cadre du deuxième projet, une cellule spectroélectrochimique contenant une grille d’or a été adaptée pour étudier des protéines redox actives. Cette grille d’or sert à la fois de substrat SERS et d’électrode de travail. Cyt c, Hb et Mb ont d'abord été utilisés pour valider la configuration, puis l'approche a été étendue pour étudier une protéine membranaire
This thesis concerns the development of surface-enhanced infrared and Raman spectroscopic approaches: surface-enhanced infrared absorption spectroscopy (SEIRAS) combined with perfusion cell and surface-enhanced Raman spectroscopy (SERS) combined with electrochemistry. Within the first project different proteins were studied: Lactose Permease (LacY), complex I and IM30.The pKa of Glu325 in LacY WT and in different mutants carrying mutations in the proton translocation active center was determined. WT complex I was oxidized with different oxidizing agents and reduced with NADH. Corresponding redox-induced conformational changes were studied. The evidence was given that Mg2+ ions induce conformational changes in the protein IM30.Within the second project the spectroelectrochemical cell containing gold grid electrode was adopted for the studies of redox active proteins. This gold grid serves both as working electrode and as SERS active substrate. First Cyt c, Hb and Mb were used to validate the setup and then the approach was extended to study a membrane protein

Thesis (Ph. D.)--University of Wisconsin--Madison, 1985.
Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 366-372).

This study aimed to obtain 15% total solids and reduced mineral content in milk UF permeate by nanofiltration, hydrolysing the lactose content of nano-concentrate enzymically, partially substituting sucrose in ice cream formulations with hydrolysed lactose nano-concentrate (HLNC), and investigating the effects of HLNC on the physio-chemical and sensory characteristics of ice cream. The desired 15% total solids in the nano-concentrate was achieved after three fold concentration of milk UF permeate. The colour of milk permeate changed, pH and mineral content decreased, and crude protein content, lactose content and titratable acidity increased. The lactose content was hydrolysed by enzyme lactase. HLNC was used to replace 25% and 50% of sucrose in ice cream formulations. Springiness, cohesiveness, chewiness, adhesiveness, hardness, iciness, Ph and colour were not significantly affected. Viscosity, freezing point, glass transition temperature, melting temperature, gumminess and sweetness were significantly decreased, whereas freezing time, saltiness and cooked flavour were significantly increased. The overall acceptability of ice cream significantly decreased at 50% but was insignificantly affected at the 25% level. These results indicate that about one quarter of sucrose could be replaced by HLNC.

Thesis (M.S.)--University of Wisconsin--Madison, 1991.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 241-247).

Doctor of Philosophy
Food Science Institute
Jayendra K. Amamcharla
Lactose is commercially produced from whey, whey permeate, or milk permeate as α-lactose monohydrate in crystalline form. Focused Beam Reflectance Measurement (FBRM) as a potential tool for in situ monitoring of lactose crystallization at concentrations relevant to the dairy industry was evaluated. Applicability of FBRM at supersaturated lactose concentrations 50%, 55%, and 60% (w/w) was reported in comparison with Brix values obtained from a Refractometer during isothermal crystallization at temperatures 20ºC and 30ºC. FBRM technique was shown to be a valuable tool for monitoring chord length distributions during lactose crystallization. In a different study, the influence of cooling rate during crystallization of lactose in concentrated permeates was studied. Three cooling rates accounting for approximately 17, 11, and 9 h were applied during lactose crystallization to evaluate the lactose crystal yield and quality of lactose crystals. There was no significant difference (P>0.05) found in lactose crystal yield, mean particle size obtained at the end of crystallization. This study suggested that increasing the cooling rate during lactose crystallization within the range explained in this study can save approximately 8 h of crystallization time. These studies evaluated FBRM as a potential tool to monitor lactose crystal chord lengths and counts. Also, process improvements were suggested to increase the productivity of lactose crystallization process by reducing the crystallization time. In chapters 5 and 6, calcium-reduced milk protein concentrates (MPCs) were used as an ingredient to improve the stability of high-protein dairy beverages. Heat stability increased significantly (P>0.05) in 8% protein solutions made from 20% calcium-reduced MPC. A significant increase in heat stability was observed in beverages formulated with 20% calcium-reduced MPC in the absence of chelating agent. In another study, it was evident that the dairy beverage formulation with 20% calcium-reduced MPC showed no sedimentation and age gelation indicating an improved storage stability. These studies confirmed that 20% calcium reduced MPC contributed towards improved heat stability and storage stability of the high-protein beverages.