Generation and characterisation of biologically active milk-derived protein and peptide fractions

In recent years, extensive research has been carried out on the health benefits of milk proteins and peptides. Biologically active peptides are defined as specific protein fragments which have a positive impact on the physiological functions of the body; such peptides are produced naturally in vivo, but can also be generated by physical and/or chemical processes, enzymatic hydrolysis and/or microbial fermentation. The aims of this thesis were to investigate not only the traditional methods used for the generation of bioactive peptides, but also novel processes such as heat treatment, and the role of indigenous milk proteases, e.g., in mastitic milk, in the production of such peptides. In addition, colostrum was characterised as a source of bioactive proteins and peptides. Firstly, a comprehensive study was carried out on the composition and physical properties of colostrum throughout the early-lactation period. Marked differences in the physico-chemical properties of colostrum compared with milk were observed. Various fractions of colostrum were also tested for their effect on the secretion of pro- and anti-inflammatory cytokines from a macrophage cell line and bone marrow dendritic cells, as well as insulin secretion from a pancreatic beta cell line. A significant reduction in the secretion of the pro-inflammatory cytokines, TNF-α, IL-6, IL-1β and IL-12, a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, as well as a significant increase in insulin secretion were observed for various colostrum fractions. Another study examined the early proteomic changes in the milk of 8 cows in response to infusion with the endotoxin lipopolysaccharide (LPS) at quarter level in a model mastitic system; marked differences in the protein and peptide profile of milk from LPS challenged cows were observed, and a pH 4.6-soluble fraction of this milk was found to cause a substantial induction in the secretion of IL-10 from a murine macrophage cell line. Heat-induced hydrolysis of sodium caseinate was investigated from the dual viewpoints of protein breakdown and peptide formation, and, a peptide fraction produced in this manner was found to cause a significant increase in the secretion of the anti-inflammatory cytokine, IL-10, from a murine macrophage cell line. The effects of sodium caseinate hydrolysed by chymosin on the gut-derived satiety hormone glucagon-like peptide-1 (GLP-1) were investigated; the resulting casein-derived peptides displayed good in vitro and in vivo secretion of GLP-1. Overall, the studies described in this thesis expand on current knowledge and provide good evidence for the use of novel methods for the isolation, generation and characterisation of bioactive proteins and/or peptides.

LIQUID-CHROMATOGRAPHY THERMOSPRAY MASS-SPECTROMETRIC ASSAY FOR TRENBOLONE IN BOVINE BILE AND FECES

A liquid chromatography-thermospray mass spectrometric assay was developed and validated to confirm the presence of illegal residues of the synthetic androgenic growth promoter, trenbolone acetate, in cattle. The assay was specific for 17alpha-trenbolone, the major bovine metabolite of trenbolone acetate. Methods were developed for the determination of 17alpha-trenbolone in both bile and faeces, the most appropriate matrices for the control of trenbolone acetate abuse. The clean-up.procedure developed relied on enzymatic hydrolysis, followed by sequential liquid-liquid and liquid-solid extraction. The extracts were then subjected to immunoaffinity chromatography. 17alpha-Trenbolone was detected by selected ion monitoring at m/z 271 using positive ion thermospray ionisation. The limit of detection was approximately 0.5 ng/g in faeces and 0.5 ng/ml in bile.

Estratégias de alteração da funcionalidade de proteínas de soja

Este trabalho teve como principal objetivo estudar e modificar as propriedades funcionais das proteínas de soja de forma a otimizar e diversificar a sua aplicação industrial. Para tal, foram propostas e estudadas quatro estratégias: i) extração do isolado de proteínas de soja (IPS) a partir de diferentes matérias-primas, ii) adição de galactomananas (GM) com graus de ramificação e massas moleculares diferentes, iii) hidrólise enzimática controlada das proteínas de soja, iv) processamento por alta pressão hidrostática. O estudo e a interpretação da influência destas estratégias sobre as propriedades funcionais das proteínas de soja, nomeadamente, na capacidade gelificante e emulsionante, foram realizados recorrendo fundamentalmente a ensaios reológicos dinâmicos a baixas deformação, espectroscopia de infravermelho, electroforeses, calorimetria diferencial de varrimento e ensaios de microscopia confocal de varrimento laser. O estudo da extração e caracterização dos isolados de proteínas de soja obtidos a partir de diferentes matérias-primas permitiu concluir que as caraterísticas físico-químicas dos isolados são dependentes da origem da matéria-prima de extração e da severidade dos tratamentos industriais prévios à extração do isolado. Contudo, as propriedades viscoelásticas dos géis obtidos por aquecimento controlado não foram significativamente distintas embora tenha sido possível relacionar o grau de agregação com a diminuição da temperatura de gelificação e com o aumento inicial dos módulos viscoelásticos. As alterações sofridas pelos isolados de origem comercial mostraram ser irreversíveis resultando em géis menos rígidos e com maior caráter viscoso. A adição de galactomanana alterou significativamente o mecanismo de gelificação induzido termicamente das proteínas de soja, bem como as propriedades viscoelásticas dos géis e a microestrutura dos géis, demonstrando-se a ocorrência de separação de fases, em virtude da incompatibilidade termodinâmica entre os biopolímeros, resultando em géis mais rígidos e no decréscimo da temperatura de gelificação. A extensão destas alterações foi dependente da massa molecular, grau de ramificação e da razão IPS/GM. O efeito da hidrólise enzimática por ação da bromelina, nas propriedades gelificantes e emulsionantes das proteínas de soja, mostrou ser dependente do grau de hidrólise (GH). Valores de GH inferiores a 15 % melhoraram as propriedades gelificantes das proteínas de soja. Por outro lado, o aumento do GH teve um efeito negativo nas propriedades emulsionantes, o qual foi atenuado por adição da goma de alfarroba, com efeito positivo na gelificação das proteínas de soja. A concentração crítica limite de compatibilidade entre os hidrolisados de proteína de soja e a goma de alfarroba aumentou com o decréscimo do GH e da massa molecular do polissacacrídeo. O efeito da AP sobre as propriedades físico-químicas e funcionais dos IPS foi influenciado pela origem do isolado e pelas condições de tratamento. O processamento até 100 MPa desencadeou um aumento da atividade emulsionante e considerável melhoria da capacidade gelificante. Contudo, valores de pressão superiores promoveram a desnaturação das proteínas constituintes dos isolados, resultando no decréscimo da temperatura de gelificação e numa re-associação das subunidades proteicas, diminuindo a elasticidade dos géis finais. Os resultados sugeriram que as alterações nas proteínas de soja promovidas durante o tratamento por AP constituem um fator limitante para o desdobramento e re-associação durante o aquecimento térmico, necessários para a formação e fortalecimento de gel formado. O processamento por AP influenciou a estrutura secundária e a microestrutura das amostras. A presença de GA teve um papel baroprotetor. Assim, com este trabalho demonstrou-se que com as estratégias seguidas para manipulação das propriedades funcionais de proteínas de soja, nomeadamente através da adição de um polissacarídeo com propriedades estruturais controladas, da adequada combinação da adição de um polissacarídeo neutro com a hidrólise controlada das proteínas ou com tratamento por alta pressão, é possível a criação de novas funcionalidades, com utilidade no desenvolvimento de novas formulações alimentares, permitindo expandir a aplicação destas proteínas vegetais.

Improved extraction of arabinoxylans from rye bran and production of health beneficial oligosaccharides

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Synthese und Charakterisierung neuer Stärkederivate für die klinische Anwendung

Ein neuartiges, mehrstufiges Syntheseverfahren wurde zur Darstellung von unterschiedlichen Stärkederivaten (Ether und Ester) entwickelt, die hinsichtlich ihres Eigenschaftsprofils und ihrer Reinheit für die klinische Anwendung als Blutvolumenersatzmittel geeignet sind. Die Synthesen wurden dahingehend gestaltet, dass Produkte mit einer hohen Regioselektivität resultierten. Dabei konnten sämtliche Reaktionsschritte, die zur Modifikation der ursprünglich eingesetzten Wachsmais- und Kartoffelstärken notwendig waren, in einem homogenen, wäss-rigen System ohne zwischenzeitliche Aufarbeitung in einem Eintopfverfahren durchgeführt werden. Die auf diese Weise bevorzugt synthetisierten Carboxymethylstärken wurden mit NMR-spektroskopischen Methoden und GPC-MALLS strukturell eingehend charakterisiert. Mit eigens entwickelten Enzym-Assays konnten essentielle Informationen über die physiolo-gische Wirksamkeit der verschiedenen Stärkederivate in vitro gewonnen werden. Die Ergebnisse konnten mit Untersuchungen an Humanblut verifiziert werden.

Monatin and its stereoisomers: chemoenzymatic synthesis and taste properties

The sweet natural compound monatin 1 has two stereogenic centres and the (2S,4S) absolute configuration has been attributed to the natural isomer. We obtained all four stereoisomers as pure compounds by a six-step synthetic sequence. The stereogenic centre at C-4 was introduced stereoselectively by a regio- and enantiospecific enzymatic hydrolysis of the racemic ethyl dicarboxylate 4 using a protease from Aspergillus oryzae. The absolute configuration of the intermediate products was assigned by X-ray diffraction of chiral derivatives. The stereogenic centre at C-2 was introduced non-specifically, and the resulting diastereomeric mixtures were separated by RP-HPLC. The absolute configurations of the final products were established by comparing retention times on a chiral HPLC column with those of known samples. The four stereoisomers were submitted to tasting trials and three of them, particularly the (2R,4R) isomer, were found to be intensely sweet. A sample of natural monatin analysed under the same conditions is shown to contain all the four stereoisomers. The relative stereoisomeric content in the plant, as well as the possible isomerisation of the chiral centres during extraction and manipulation of monatin samples, are important points that need to be clarified by extensive analysis of the natural extracts. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

Pectins and pectic-oligosaccharides inhibit Escherichia coli O157 : H7 Shiga toxin as directed towards the human colonic cell line HT29

Pectins and pectic-oligosaccharides, as derived by controlled enzymatic hydrolysis, were evaluated for their ability to interfere with the toxicity of Shiga-like toxins from Escherichia coli O157:H7. Both types of material resulted in some degree of protection but this was significantly higher (P > 0.01) with the oligosaccharide fractions (giving 90-100% cell survival, compared to 70-80% with the polymer). An effect of methylation on the protective effect was detected with lower degrees being more active. The pectic-oligosaccharides and galabiose, the minimum toxin receptor analogue, were shown to inhibit toxicity and were both protective at 10 mg ml(-1), but not at lower concentrations. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Bioactive peptides from food proteins: new opportunities and challenges

Food proteins such as milk and soy are a rich source of bioactive peptides. In the last decade, research into this area has intensified and new bioactive peptide sequences have been discovered with a range of apparent biological functions; for example, antihypertensive, antioxidant, and antimicrobial effects and opiate-like qualities have been reported. These peptides could therefore lead to the development of important functional food products and ingredients for the prevention and even treatment of chronic diseases such as cardiovascular disease and cancer. Peptides can be produced by fermentation with dairy starters for instance, and by enzymatic hydrolysis with pancreatic and microbial enzymes. Further purification is typically carried out by membrane filtration and/or chromatographic methods. The production of novel bioactive peptides and their incorporation into functional food products poses several technological challenges as well as regulatory and marketing issues. Proof of efficacy is of paramount importance; this should be verified by conducting appropriate tests in vivo in animals and in humans. In addition, tests for cytotoxicity and allergenicity must be conducted. Despite all of these hurdles, scientific evidence is increasingly demonstrating the health benefits of diet-based disease prevention, and therefore new developments in this area are likely to continue both at the research and the commercialisation level.

Functional oligosaccharides: application and manufacture.

Oligosaccharides are attracting increasing interest as prebiotic functional food ingredients. They can be extracted or obtained by enzymatic hydrolysis from a variety of biomass sources or synthesized from simple oligosaccharides by enzymatic transfer reactions. The major prebiotic oligosaccharides on the market are inulin, fructo-oligosaccharides, and galacto-oligosaccharides. They have been evaluated using a range of in vitro and in vivo methods, although there is a need for more large-scale human trials using modern microbiological methods. Prebiotics are being studied for their effects on gut health and well being and specific clinical conditions, including colon cancer, inflammatory bowel disease (IBD), acute infections, and mineral absorption. Developing understanding of the functional ecology of the human gut is influencing current thinking on what a prebiotic might achieve and is providing new targets for prebiotic intervention.

Bioethanol from lignocelluloses: Status and perspectives in Brazil

The National Alcohol Program - ProAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse. (C) 2009 Elsevier Ltd. All rights reserved.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Hidrólise de resíduos lignocelulósicos utilizando extrato enzimático celulolítico produzido por Trichoderma reesei atcc 2768

Cellulolytic enzymatic broth by Trichoderma reesei ATCC 2768 cultived in shaker using cashew apple bagasse and coconut shell bagasse, as substrate for fermentation, was used to investigate the enzymatic hydrolysis of these substrates after pre-treatment with 1 M NaOH, wet-oxidation as well as a combination of these treatments. Hydrolysis runs were carried at 125 rpm, 50ºC and initial pH of 4.8 for 108 hours. Enzymatic broth produced using cashew apple bagasse treated with 1M NaOH (1.337 UI/mL CMCase and 0.074 UI/mL FPase), showed after the hydrolysis an initial of 0.094 g of reducing sugar/g of substrate.h with 96% yield of total reducing sugars while for the coconut shell bagasse treated using the alkaline process (0.640 UI/mL CMCase and 0.070 UI/mL FPase) exhibited an initial hydrolysis velocity of 0.025 g of reducing sugar/g of substrate.h with 48% yield of total reducing sugars. For the treatment with wet-oxidation using cashew apple bagasse as substrate enzymatic broth (0.547 UI/mL CMCase) exhibited an initial hydrolysis velocity of 0.014 g of reducing sugars/g of substrate.h with a lower yield about 89% of total reducing sugars compared to the alkaline treatment. Enzymatic broth produced using coconut shell treated by wet-oxidation showed an initial hydrolysis velocity of 0.029 g of reducing sugar/g of substrate.h with 91% yield. However, when the combination of these two treatments were used it was obtained an enzymatic broth of 1.154 UI/mL CMCase and 0.107 FPase for the cashew apple bagasse as well as 0.538 UI/mL CMCase and 0,013 UI/mL de FPase for the coconut shell bagasse. After hydrolysis, initial velocity was 0.029 g of reducing sugar/g of substrate.h. with 94% yield for the cashew apple bagasse and 0.018 g de reducing sugar/g of substrate.h with 69% yield for coconut shell bagasse. Preliminary treatment improves residues digestibility showing good yields after hydrolysis. In this case, cellulose from the residue can be converted into glucose by cellulolytic enzymes that can be used for ethanol production

Estudo do mecanismo de produção de oligossacarídeos com atividades nutracêuticas a partir da quitosana por hidrólise enzimática com processo fermentativo simultâneo

The obtaining of the oligosaccharides from chitosanase, has showed interest of the pharmaceutical area in the last years due their countless functional properties. Although, the great challenge founded out is how to keep a constant and efficient production. The alternative proposed by this present work was to study the viability to develop an integrated technology, with reduced costs. The strategy used was the obtaining of the oligomers through enzymatic hydrolysis using chitosanolitic enzymes obtained straight from the fermented broth, eliminating this way the phases involved in the enzymes purification. The two chitosanases producing strains chosen for the work, Paenibacillus chitinolyticus and Paenibacillus ehimensis, were evaluated according to the behavior in the culture medium with simple sugar and in relation to the pH medium variations. The culture medium for the chitosanases induction and production was developed through addition of soluble chitosan as carbon source. The soluble chitosan was obtained using hydrochloric acid solution 0.1 M and afterwards neutralization with NaOH 10 M. The enzymatic complexes were obtained from induction process in culture medium with 0.2% of soluble chitosan. The enzymes production was verified soon after the consumption of the simple sugars by the microorganisms and the maximum chitosanolitic activity obtained in the fermented broth by Paenibacillus chitinolyticus was 249 U.L-1 and by Paenibacillus ehimensis was 495U.L-1. These two enzymatic complexes showed stability when stored at 20°C for about 91 days. The enzymes in the fermented broth by Paenibacillus chitinolyticus, when exposed at temperature of 55°C and pH 6.0, where the activity is maximum, showed 50% lost of activity after 3 hours Meanwhile, for the complex produced by Paenibacillus ehimensis, after 6 days of exposure, it was detected 100% of the activity. The chito-oligosaccharides obtained by the hydrolysis of a 1% chitosan solution, using the enzymatic complex produced by Paenibacillus chitinolyticus showed larger quantity after 9 hours hydrolysis and using the complex produced by Paenibacillus ehimensis after 20 minutes was observed the chito-ligosacharides with polymerization degree between 3 and 6 units. Evaluating these results, it was verified that the production of chitosan-oligosaccharides is possible, using a simultaneous process

Produção de bioetanol a partir de pedúnculo de caju (Anacardium occidentale L.) por fermentação submersa

Recently, global demand for ethanol fuel has expanded very rapidly, and this should further increase in the near future, almost all ethanol fuel is produced by fermentation of sucrose or glucose in Brazil and produced by corn in the USA, but these raw materials will not be enough to satisfy international demand. The aim of this work was studied the ethanol production from cashew apple juice. A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentration (from 24.4 to 103.1 g.L-1). Maximal ethanol, cell and glycerol concentrations (44.4 g.L-1, 17.17 g.L-1, 6.4 g.L-1, respectively) were obtained when 103.1 g.L-1 of initial sugar concentration were used, respectively. Ethanol yield (YP/S) was calculated as 0.49 g (g glucose + fructose)-1. Pretreatment of cashew apple bagasse (CAB) with dilute sulfuric acid was investigated and evaluated some factors such as sulfuric acid concentration, solid concentration and time of pretreatment at 121°C. The maximum glucose yield (162.9 mg/gCAB) was obtained by the hydrolysis with H2SO4 0.6 mol.L-1 at 121°C for 15 min. Hydrolysate, containing 16 ± 2.0 g.L-1 of glucose, was used as fermentation medium for ethanol production by S. cerevisiae and obtained a ethanol concentration of 10.0 g.L-1 after 4 with a yield and productivity of 0.48 g (g glucose)-1 and 1.43 g.L-1.h-1, respectively. The enzymatic hydrolysis of cashew apple bagasse treated with diluted acid (CAB-H) and alkali (CAB-OH) was studied and to evaluate its fermentation to ethanol using S. cerevisiae. Glucose conversion of 82 ± 2 mg per g CAB-H and 730 ± 20 mg per g CAB-OH was obtained when was used 2% (w/v) of solid and loading enzymatic of 30 FPU/g bagasse at 45 °C. Ethanol concentration and productivity was achieved of 20.0 ± 0.2 g.L-1 and 3.33 g.L-1.h-1, respectively when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g.L-1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g.L-1), ethanol concentration and productivity was 8.2 ± 0.1 g.L-1 and 2.7 g.L-1.h-1, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 g/g glucose and 0.47 g/g glucose, with pretreated CABOH and CAB-H, respectively. The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated too in this work. First, the yeast CE025 was preliminary cultivated in a synthetic medium containing glucose and xylose. Results showed that it was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pre-treatment. The fermentation of CABH was conducted at pH 4.5 in a batch-reactor, and only ethanol was produced by K. marxianus CE025. The influence of the temperature in the kinetic parameters was evaluated and best results of ethanol production (12.36 ± 0.06 g.L-1) was achieved at 30 ºC, which is also the optimum temperature for the formation of biomass and the ethanol with a volumetric production rate of 0.25 ± 0.01 g.L-1.h-1 and an ethanol yield of 0.42 ± 0.01 g/g glucose. The results of this study point out the potential of the cashew apple bagasse hydrolysate as a new source of sugars to produce ethanol by S. cerevisiae and K. marxianus CE025. With these results, conclude that the use of cashew apple juice and cashew apple bagasse as substrate for ethanol production will bring economic benefits to the process, because it is a low cost substrate and also solve a disposal problem, adding value to the chain and cashew nut production

Xylanase production by Aspergillus versicolor

The xylanolytic system of Aspergillus versicolor is controlled by induction and carbon catabolite repression. Carboxymethylcellulose and wheat bran were the best inducers of xylanolytic activity. When the fungus was grown for 5 days on VOGEL's liquid medium with wheat bran, the optimal pH and temperature for xylanase production were 6.5 and 30 degrees C, respectively. Optimal conditions for the xylanolytic activity assay were at pH 6.0 and 55 degrees C. The half-life at 60 degrees C of the crude enzyme was 6.5 and 21 minutes, in the absence or presence of substrate, respectively.Xylan is the main hemicellulosic component of plant biomass being present in appreciable quantities in agricultural and several agroindustrial wastes. From the products of xylan enzymatic hydrolysis it is possible to obtain cell protein, fuels and other chemicals. Xylanases combined with cellulase could have applications in food processing. Cellulase-free xylanases can be also utilized for preparation of cellulose pulps and liberation of textile fibres (WOODWARD 1984; BIELY 1985, WONG et al. 1988). In view of the potential applications of xylanases, a study of these enzymes from various sources and their multiplicity is desirable.Among xylanolytic microorganisms, filamentous fungi have been more extensively studied and the genus Aspergillus has been shown to be an efficient producer of xylanases. Preliminary observations from our laboratory have demonstrated that a strain of Aspergillus versicolor, isolated from Brazilian soil, produced high xylanase and low cellulase levels, which is an interesting characteristic for some industrial applications. In this report we describe the production and some properties of xylanase obtained from this fungus.