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Diatoms are currently considered valuable feedstocks for different biotechnological applications. To deepen the knowledge on the production of these microalgae, the diel pattern of batch growth, photosystem II performance, and accumulation of target metabolites of two commercially relevant diatoms, Phaeodactylum tricornutum and Skeletonema costatum, were followed outdoors in 100-L flat panel photobioreactors. S. costatum presented a higher light-to-biomass conversion resulting in higher growth than P. tricornutum. Both fluorescence data and principal component analysis pointed to temperature as a limiting factor for the growth of P. tricornutum. Higher protein and carbohydrate contents were found in P. tricornutum, whereas S. costatum fatty acids were charac-terized by a higher unsaturation degree. Higher productivities were found at 1 p.m. for protein, lipid, and ash in the case of S. costatum. Overall, S. costatum showed great potential for outdoor cultivation, revealing a broader temperature tolerance and increased biomass productivity than P. tricornutum. info:eu-repo/semantics/publishedVersion

Few studies have focused on the biotechnological potential of the marine cyanobacterium Synechocystis salina, in particular its bioactive metabolites. Hence, an opportunity exists to explore various solvent combinations in attempts to improve extraction of cellular contents of metabolites with antioxidant features as a part of a structured attempt to determine whether S. salina LEGE 06,155 holds a promise for industrial exploitation. Extracts were obtained by single extractions with four GRAS solvents: hexane (H), acetone (A), ethanol (E), and buffer phosphate (PBS) (series A) and successive extractions: H-E-PBS (series B), H-A-PBS (series C), and H-A-E-PBS (series D). The yields, antioxidant capacity, and cytotoxicity of the extracts, as well their content in pigments, lipids, and phenolic compounds were assessed. The combination of solvents of series B produced the highest yield of extraction, of 33.56%. Aqueous extracts for single (series A) and successive extractions (series B and C) presented the highest antioxidant capacity, for ABTS•+ and NO•−, respectively, and had a high content of phycobiliproteins (PBPs). The acetonic (series A) and ethanolic (series B) extracts also showed high antioxidant capacity, in particular for DPPH• and O2•−, respectively. Overall, organic extracts did not show signs of cytotoxicity. Series B extraction enhanced general pigment content, yet single extraction with ethanol (series A) produced the highest content in carotenoids, in particular echinenone and β-carotene, and PBS (series A) improved PBPs content. The lipid content was enhanced in series A (ethanol), while the total phenolic content was improved in series B. Synechocystis has accordingly demonstrated potential for eventual exploitation as biotechnological resource, in terms value-added compounds with antioxidant features.

A cyanobacterium of the genus Chroococcidiopsis has been recently unveiled to go to Mars, owing to its unique ability to stand harsh environmental conditions. Nevertheless, it has been little characterized in terms of other biotechnological applications. The aim of this study was thus to explore the biotechnological potential of Chroococcidiopsis sp. LEGE 06174, using GRAS (generally recognized as safe) solvents for the combined extraction of bioactive compounds. Due to its mucilaginous sheath, sulfuric acid, NaOH, PBS, DMSO 20%, DMSO 100%, and acetone were tested as preliminary treatment—in attempts to enhance pigment extraction. Extracts were then obtained by several series of successive extractions: phosphate-buffered saline (PBS) (serie A), ethanol-PBS (serie B), acetone-PBS (serie C), methanol-PBS (serie D), and acetone-methanol-PBS (serie E). Such extracts were screened for antioxidant capacity, cytotoxicity, and physicochemical characterization in terms of polysaccharides, pigments, and total phenolic compounds. Pre-treatment with PBS proved the most effective toward overall pigment extraction and dissolution of the said mucilaginous sheath. Chroococcidiopsis sp. extracts have unfolded some interesting bioactive features, particularly upon pre-treatment with PBS—i.e., high antioxidant capacity and high content in polysaccharides, scytonemin, phycobiliproteins, and phenolic compounds. The highest total pigment content was obtained in serie D, particularly carotenoids, whereas the highest level of phenolic compounds was found in serie B. All in all, Chroococcidiopsis sp. appears to constitute a significant biotechnological resource worthy of further exploitation in terms of high added-value compounds.

Microalgae have shown a great potential as a source of bioactive compounds, such lipidic compounds as fatty acids and carotenoids, with recognized benefit for human health. Their synthesis is directly affected by culturing conditions, but, as photosynthetic organisms, microalgae are greatly influenced by light, particularly light quality and irradiance, responsible for growth regulation and activation of biochemical pathways. Therefore, the aim of this work was to maximize biomass and high added-value compound productivity by Scenedesmus obliquus M2-1, using different light irradiances of two previously selected light sources for the production of antioxidant compounds: monochromatic red light (R) and a mixture of 40% of blue and 60% of red light (BR). Four irradiances (50, 100, 150, and 200 μmolphotons m−2 s−1) were accordingly evaluated for both light sources, upon productivity of biomass, fatty acids, carotenoids, and total antioxidant compounds. Changes in light quality and irradiance influenced these aforementioned productivities. Optimal condition for biomass productivity was 150 μmolphotons m−2 s−1, regardless light quality, for both sources. On the other hand, when the production targets carotenoids, polyunsaturated fatty acids, and/or total antioxidant capacity, the optimal condition was found to be at 200 μmolphotons m−2 s−1 under BR light.

Light plays a crucial role in photosynthetic microalgae, and its quality is a key point for biomass production and the variability of the organisms’ biochemical composition, with blue and red, the light bands being the most effectively absorbed by green algae. Recently, light-emitting diodes (LEDs) have emerged as an alternative that allow a better modulation of algal metabolism. The aim of this work was to study the effects of blue and red LEDs on antioxidant capacity, biomass productivity (Px) and carotenoid and fatty acid content over time in a microalga known to be a producer of antioxidant compounds, Scenedesmus obliquus M2-1. Monochromatic blue (B) and red (R) LEDs and two dichromatic combinations of blue and red (BR (40:60) and BR (50:50)) were tested, and fluorescent lamp (FL) was used as a control. Then, supplementation effects of near-infrared radiation (NIR) LEDs were assessed in the same in parameters. Results show that B induced higher PX and higher content in fatty acids when compared with other LED conditions tested. Scenedesmus obliquus M2-1 under R attained a higher antioxidant capacity and under BR (40:60) a higher carotenoid production, particularly lutein and β-carotene, when compared with that of FL. NIR supplementation in BR (40:60) and R treatments enhanced PX and fatty acid content, changing the profile of antioxidant compound production over time, but keeping the maximum concentration. Additionally, NIR supplementation decreased the total carotenoid production. This study reveals that the use of R and BR (40:60) LEDs are a useful tool to enhance the production of carotenoids in S. obliquus M2-1.

A laboratory experiment is described in which students carry out immobilization of the enzyme invertase via entrapment in alginate (Ca-alginate) and, accordingly, estimate values for kinetic consta...

The application of microalgae in wastewater treatment has recently been at the forefront of interest due to the increasing concern about environmental protection and economic sustainability. This work aimed to study two chlorophyte species, Chlorella vulgaris and Scenedesmus acutus, co-cultured outdoors in centrate of municipal wastewater as a nutrient source. Two different thin-layer units were used in these trials & mdash; thin-layer cascade (TLC) and thin-layer raceway pond (TL-RWP), suitable for this purpose due to their high biomass productivity and better culture transparency when using muddy wastewater. The units were operated in batch, and subsequently in semi-continuous growth regime & mdash; and monitored in terms of photosynthetic performance, growth, nutrient removal rate, and bioactivity. The results showed that the co-cultures grew well in the centrate, achieving the maximum biomass densities of 1.3 and 2.1 g DW L-1 in TLC and TL-RWP, respectively, by the end of the batch regime and 1.9 and 2.0 g DW L-1 by the end of the semi-continuous regime. Although TL-RWP grown cultures showed faster growth, the TLC-one revealed better nutrient removal efficiencies batch wise than the culture grown in TL-RWP & mdash; removing up to 48% of total nitrogen and 43% of total phosphorus. Conversely, the latter was more efficient under the semi-continuous regime (54% and 42% consumption of total nitrogen and phosphorus, respectively). In the harvested biomass, an important antimicrobial activity (specifically antifungal) was detected. In this sense, the in-vitro growth of the oomycete Pythium ultimum was inhibited by up to 45% with regard to the control. However, no biostimulating activity was observed. The present findings confirm the possibility of using these two species for biomass production in municipal wastewater centrate using highly productive thin-layer systems. This technology can be a valuable contribution to circular economy since the produced biomass can be re-applied for agricultural purposes. SFRH/BD/129952/2017; UIDB/00511/2020; DINOSSAUR-PTDC/BBB-EBB/1374/2014-POCI-01-0145-FEDER-016640; info:eu-repo/semantics/publishedVersion

Tisochrysis lutea is a marine haptophyte rich in omega-3 polyunsaturated fatty acids (e.g., docosahexaenoic acid (DHA)) and carotenoids (e.g., fucoxanthin). Because of the nutraceutical applications of these compounds, this microalga is being used in aquaculture to feed oyster and shrimp larvae. In our earlier report, T. lutea organic crude extracts exhibited in vitro cytotoxic activity against human hepatocarcinoma (HepG2) cells. However, so far, the compound(s) accountable for the observed bioactivity have not been identified. Therefore, the aim of this study was to isolate and identify the chemical component(s) responsible for the bioactivity observed. Bioassay-guided fractionation through a combination of silica-gel column chromatography, followed by preparative thin layer chromatography (PTLC), led to the isolation of two diastereomers of a monoterpenoid lactone, namely, loliolide (1) and epi-loliolide (2), isolated for the first time in this species. The structural elucidation of both compounds was carried out by GC-MS and 1D (1H and 13C APT) and 2D (COSY, HMBC, HSQC-ed, and NOESY) NMR analysis. Both compounds significantly reduced the viability of HepG2 cells and were considerably less toxic towards a non-tumoral murine stromal (S17) cell line, although epi-loliolide was found to be more active than loliolide. FCT, Portugal: UID/Multi/04326/2019, UID/MAR/04292/2020 (MARE), UID/Multi/04046/2020 (BioISI) and from the grant of 0055 ALGARED +5E—INTERREG V-A; SFRH/BD/116604/2016; SFRH/BD/105541/2014 info:eu-repo/semantics/publishedVersion

Since temperature is an important factor affecting microalgal growth, photosynthetic rate and biomass composition, this study has accordingly focused on its effects on biomass yield and nighttime biomass loss, as well as photochemical changes, using Nannochloropsis oceanica as model species, grown in two outdoor 50-L tubular photobioreactors (PBR). In two independent trials, cultures were subjected to a diurnal light:dark cycle, under a constant temperature of 28 degrees C and, on the second trial, at 18 degrees C. Changes in culture performance were assessed by measuring growth, lipid and fatty acid composition of the biomass in both morning and evening. Our results revealed that N. oceanica shows a wide temperature tolerance with relevant nighttime biomass loss, that decreased at lower temperatures, at the expenses of its daily productivity. Fluorescence measurements revealed reversible damage to photosystem II in cells growing in the PBR under optimal thermal conditions, whereas microalgae grown at suboptimal ones exhibited an overall lower photosynthetic activity. Lipids were partially consumed overnight to support cell division and provide maintenance energy. Eicosapentaenoic acid (EPA) catabolism reached a maximum after the dark period, as opposed to their saturated counterparts; whereas lower temperatures led to higher EPA content which reached the maximum in the morning. These findings are relevant for the production of Nannochloropsis at industrial scale. European Cooperation in Science and Technology (COST) Action: European network for algal-bio-products (EUALGAE) [ES1408] Portuguese national funds from the Foundation for Science and Technology (FCT) [SFRH/BD/129952/2017] Laboratory for Process Engineering, Environment, Biotechnology and Energy -LEPABE -by the FCT/MCTES (PIDDAC) [UIDB/00511/2020] project: "LEPABE-2-ECO-INNOVATION" - North Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) [NORTE-01-0145-FEDER-000005] project: "DINOSSAUR" - ERDF through Programa Operacional Competitividade e Internacionalizacao (COMPETE2020) [PTDC/BBB-EBB/1374/2014-POCI-01-0145-FEDER-016640] project: "SABANA"- European Union [727874] [UID/Multi/04326/2019] info:eu-repo/semantics/publishedVersion

Spontaneous fermentation by native strains of lactic acid bacteria has traditionally been used to preserve table olives, by taking advantage of a proactive and competitive reduction of growth of unwanted microorganisms. Starter cultures have proven effective in stabilizing fermentation processes at large. Despite the many starter cultures available commercially, few have originated on the native microflora – and table olives have seldom been processed via deliberate inoculation with them. This study screened variables relevant for the growth of Lactobacillus plantarum AFS13 originally obtained from brines in Alto Douro (Portugal); and for preservation thereof as the starter, should it prove appropriate. A 2 I V 6 − 2 factorial design was used to assess six parameters (and their interactions): 19 fermentation batches were carried out to ascertain growth rates. Temperature and aeration degree were the most significant processing parameters. Maximum growth rate, 0.356 h−1, was attained under anaerobic conditions, at 37 °C, and in the absence of NaCl; ca. 9 log10 CFU mL−1 was reached by 6 h. Glucose concentration, pH, and stirring rate proved not significant, so economic considerations should fix their optimum levels. The starter culture could be preserved for up to 4 mo after freeze-drying in sucrose, with an acceptable reduction in cell viability.

This research effort aimed at a better understanding of microbial phenomena taking place during time in spontaneous sourdough fermentation for broa, a traditional Portuguese bread. Unlike most microbiological studies of sourdough, viable counts obtained were not limited to Lactobacillus and yeasts, but encompassed also molds, Gram-negative rods, endospore-(non)forming Gram-positive rods and catalase-positive/negative Gram-positive cocci. This less conventional approach unveiled the ubiquitous Bacillus genus throughout spontaneous broa sourdough fermentation. Presumptive yeasts, Lactobacillus and Bacillus were found to low levels after kneading, but became dominant by the end of regular (no aeration) fermentation. They apparently inhibit undesired rods and Staphylococcus, which were found to relatively high numbers after kneading. By 24 h of fermentation, lactic acid cocci accounted for an important fraction of biodiversity, and pH decreased significantly reaching about 4.1. Aeration accelerated the microbial dynamics. In terms of total viable counts, such a long-term fermentation appeared to reach a quasi-stationary state. Practical Applications Microbial studies encompassing a large set of culture media and incubation conditions were previously undertaken in samples of maize and rye flours, and sourdoughs for broa obtained from 14 artisanal producers and in two seasons. Microbial counts and identification unfolded a complex wild microbiota, and fermentation played a major effect upon all microbial groups. Flours and mother-dough microbiota were characterized during storage afterward. A similar approach was followed here to ascertain the microbiological profile of a wide range of microorganisms throughout long-term sourdough fermentation, in attempts to answer the questions: how microorganisms evolve, and how they respond to increasing aeration. These studies provide an innovative way to look at sourdough systems and microbial dynamics therein. Results showed the importance of increasing fermentation time to take full advantage of ecological competition against undesirable microorganisms, and emphasized the need for further studies to reveal the role of Bacillus in sourdough fermentations.

The nutraceutical potential of microalgae boomed with the exploitation of new species and sustainable extraction systems of bioactive compounds. Thus, a laboratory-made continuous pressurized solvent extraction system (CPSE) was built to optimize the extraction of antioxidant compounds, such as carotenoids and PUFA, from a scarcely studied prokaryotic microalga, Gloeothece sp. Following &ldquo, green chemical principles&rdquo, and using a GRAS solvent (ethanol), biomass amount, solvent flow-rate/pressure, temperature and solvent volume&mdash, including solvent recirculation&mdash, were sequentially optimized, with the carotenoids and PUFA content and antioxidant capacity being the objective functions. Gloeothece sp. bioactive compounds were best extracted at 60 &deg, C and 180 bar. Recirculation of solvent in several cycles (C) led to an 11-fold extraction increase of &beta, carotene (3C) and 7.4-fold extraction of C18:2 n6 t (5C) when compared to operation in open systems. To fully validate results CPSE, this system was compared to a conventional extraction method, ultrasound assisted extraction (UAE). CPSE proved superior in extraction yield, increasing total carotenoids extraction up 3-fold and total PUFA extraction by ca. 1.5-fold, with particular extraction increase of 18:3 n3 by 9.6-fold. Thus, CPSE proved to be an efficient and greener extraction method to obtain bioactive extract from Gloeothece sp. for nutraceutical purposes&mdash, with low levels of resources spent, while lowering costs of production and environmental impacts.

The development of bio-based nanostructures as nanocarriers of bioactive compounds to specific body sites has been presented as a hot topic in food, pharmaceutical and nanotechnology fields. Food and pharmaceutical industries seek to explore the huge potential of these nanostructures, once they can be entirely composed of biocompatible and non-toxic materials. At the same time, they allow the incorporation of lipophilic and hydrophilic bioactive compounds protecting them against degradation, maintaining its active and functional performance. Nevertheless, the physicochemical properties of such structures (e.g., size and charge) could change significantly their behavior in the gastrointestinal (GI) tract. The main challenges in the development of these nanostructures are the proper characterization and understanding of the processes occurring at their surface, when in contact with living systems. This is crucial to understand their delivery and absorption behavior as well as to recognize potential toxicological effects. This review will provide an insight into the recent innovations and challenges in the field of delivery via GI tract using bio-based nanostructures. Also, an overview of the approaches followed to ensure an effective deliver (e.g., avoiding physiological barriers) and to enhance stability and absorptive intestinal uptake of bioactive compounds will be provided. Information about nanostructures potential toxicity and a concise description of the in vitro and in vivo toxicity studies will also be given., Joana T. Martins, Oscar L. Ramos, Ana C. Pinheiro, Ana I. Bourbon, Helder D. Silva and Miguel A. Cerqueira (SFRH/BPD/89992/2012, SFRH/BPD/80766/2011, SFRH/BPD/101181/2014, SFRH/BD/73178/2010, SFRH/BD/81288/2011, and SFRH/BPD/72753/2010, respectively) are the recipients of a fellowship from the Fundacao para a Ciencia e Tecnologia (FCT, POPH-QREN and FSE, Portugal). The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the project "BioInd-Biotechnology and Bioengineering for improved Industrial and Agro-Food processes," REF.NORTE-07-0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. We also thank to the European Commission: BIOCAPS (316265, FP7/REGPOT-2012-2013.1) and Xunta de Galicia: Agrupamento INBIOMED (2012/273) and Grupo con potencial de crecimiento. The support of EU Cost Action FA1001 is gratefully acknowledged.

This work attempted to assess the effect of high intensity ultrasound (HIUS) upon development of bio-based delivery systems, from β-lactoglobulin (β-Lg) gelled microparticles, for encapsulation of a bioactive peptide concentrate (PepC). Solutions of 150 g L−1 of commercial β-Lg and 30 g L−1 PepC, at various pH values (3.0, 4.0 and 5.5), were accordingly subjected to gelation for 30 min using a dry bath kept at 80 °C. The gelled systems were then exposed to HIUS at 0–4 °C, and the effect of processing time (2.5–20.0 min) was ascertained. Laser light scattering and confocal microscopy were used to characterize the particle size distribution, prior to and immediately after HIUS treatment. Gels obtained at pH 5.5 and 4.0 were harder than those obtained at pH 3.0. Ultrasound treatment of gels produced an important reduction in particle mean diameter as sonication time elapsed. Confocal microscopy indicated that application of HIUS led to almost round and monodispersed particles, at both pH 5.5 and 4.0. The peptide encapsulation efficiency was assessed by chromatography and accompanied by assay for bioactivity, after precipitation of the encapsulated material and analysis of the soluble peptides therein., Tânia Tavares and Oscar L. Ramos gratefully acknowledge Post-Doctoral grants (SFRH/BPD/89360/2012 and SFRH/BPD/80766/2011, respectively) by Fundação para a Ciência e Tecnologia (FCT, Portugal), under the supervision of author F. Xavier Malcata. Partial financial support for the research expenses was provided by CYTED Program. The authors are grateful to FCT Strategic Project PEst-OE/EQB/LA0023/2013 and Project BioEnv—Biotechnology and Bioengineering for a sustainable world, Ref. NORTE-07-0124-FEDER-000048, co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER; and Project POCI-01-0145-FEDER-006939—Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia—LEPABE and NORTE-01-0145-FEDER-000005—LEPABE-2-ECO-INNOVATION, funded by Fundo Europeu de Desenvolvimento Regional (FEDER), through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and Programa Operacional Regional do Norte (NORTE2020), and by national funds through FCT. The authors are also indebted to Nerina Camino and Ana Pilosof, from Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, for generous and stimulating support, and for their relevant contributions to this field of research., info:eu-repo/semantics/publishedVersion

Whey proteins are widely used as nutritional and functional ingredients in formulated foods because they are relative inexpensive, generally recognized as safe (GRAS) ingredient and possess important biological, physical and chemical functionalities. Denaturation and aggregation behavior of these proteins is of particular relevance toward manufacture of novel nanostructures with a number of potential uses. When these processes are properly engineered and controlled, whey proteins may be formed into nanohydrogels, nanofibrils or nanotubes and be used as carrier of bioactive compounds. This review intends to discuss the latest understandings of nanoscale phenomena of whey protein denaturation and aggregation that may contribute for the design of protein nanostructures. Whey protein aggregation and gelation pathways under different processing and environmental conditions such as microwave heating, high voltage and moderate electrical fields, high pressure, temperature, pH and ionic strength were critically assessed. Moreover, several potential applications of nanohydrogels, nanofibrils and nanotubes for controlled release of nutraceutical compounds (e.g. probiotics, vitamins, antioxidants and peptides) were also included. Controlling the size of protein networks at nanoscale through application of different processing and environmental conditions can open perspectives for development of nanostructures with new or improved functionalities for incorporation and release of nutraceuticals in food matrices., Oscar L. Ramos, Ricardo N. Pereira and Clara Fuci~nos gratefully acknowledge their Post-Doctoral grants (SFRH/BPD/80766/2011, SFRH/BPD/ 81887/2011, and SFRH/BPD/87910/2012, respectively) to the Fundação para a Ciência e Tecnologia (FCT, Portugal). All authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project “BioEnv— Biotechnology and Bioengineering for a sustainable world”, REF. NORTE07-0124-FEDER-000048, co-funded by Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER.

Pico cheese is a traditional variety manufactured on the eponymous Azorean Island (Portugal). In total, 153 cheeses were analysed. The average total solids (TS) content by the end of maturation was 63.3 +- 0.3 g/100 g, Aw 0.943-0.966, salt-in-moisture 2.12-4.17 g/100 g and pH 4.89-5.11 +-0.02. Proteolysis was similar to that of other Iberian raw milk cheeses, but the free fatty acid content was generally higher. Lactococci were the predominant bacteria. Coagulase-positive staphylococci constituted the most severe hygiene problem. Positive correlations were found between maturation time and TS, titratable acidity, Aw, proteolysis indices and Enterobacteriaceae and coagulase-positive staphylococcus viable numbers.

The present work aimed to determine the antioxidant, metal chelating and neuroprotective potential of the unsaponifiable matter (UM) of Tetraselmis chuii to be applied to a biorefinery setting. The UM obtained via saponification from crude lipids extracted from microalgal wet biomass showed a radical scavenging activity (RSA) towards the DPPH radical of 90.7 +/- 1.3% and 57.1 +/- 1.2% at a concentration of 10 and 5 mg/ mL, respectively. The UM fraction also displayed metal chelating capacity at a concentration of 5 mg/ mL: 58.5 +/- 1.4% and 50.9 +/- 4.0% for copper and iron, respectively. The chemical characterization of the UM revealed significant levels of total phenolics (TPC, 13.61 mg GAE/g) and carotenoids (2.45 mg/g of beta-carotene, lutein and violaxanthin). Overall, the separation of the UM containing high value metabolites might significantly upgrade the total wet biomass value in a biorefinery, allowing the exploitation of a stream with relevant antioxidant and metal chelating activities. SFRH/BD/105541/2014, IF/00049/2012, CCMAR/Multi/04326/2013 info:eu-repo/semantics/publishedVersion

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period., Oscar L. Ramos, Ricardo N. Pereira and Ana C. Pinheiro gratefully acknowledge their Post-Doctoral grants (SFRH/BPD/80766/2011, SFRH/BPD/81887/2011 and SFRH/BPD/101181/2014, respectively) and Ana I. Bourbon gratefully acknowledge her Doctoral grant (SFRH/BD/73178/2010) to Fundacao para a Ciencia e a Tecnologia (FCT). The authors would like to acknowledge to Francisco Figueiredo from the Institute for Molecular and Cell Biology (IBMC), University of Porto, for assistance in taking the TEM pictures. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project "BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER.

Formation of whey protein isolate protein aggregates under the influence of moderate electric fields upon ohmic heating (OH) has been monitored through evaluation of molecular protein unfolding, loss of its solubility, and aggregation. To shed more light on the microstructure of the protein aggregates produced by OH, samples were assayed by transmission electron microscopy (TEM). Results show that during early steps of an OH thermal treatment, aggregation of whey proteins can be reduced with a concomitant reduction of the heating chargeby reducing the come-up time (CUT) needed to reach a target temperatureand increase of the electric field applied (from 6 to 12 V cm1). Exposure of reactive free thiol groups involved in molecular unfolding of -lactoglobulin (-lg) can be reduced from 10 to 20 %, when a CUT of 10 s is combined with an electric field of 12 V cm1. Kinetic and multivariate analysis evidenced that the presence of an electric field during heating contributes to a change in the amplitude of aggregation, as well as in the shape of the produced aggregates. TEM discloses the appearance of small fibrillar aggregates upon the influence of OH, which have recognized potential in the functionalization of food protein networks. This study demonstrated that OH technology can be used to tailor denaturation and aggregation behavior of whey proteins due to the presence of a constant electric field together with the ability to provide a very fast heating, thus overcoming heat transfer limitations that naturally occur during conventional thermal treatments., The authors thank the Portuguese Foundation for Science and Technology (FCT) strategic project UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124FEDER-027462), and the project BBioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes^ REF. NORTE-07-0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte(ON.2–O Novo Norte), QREN, FEDER. The authors would like to acknowledge Rui Fernandes from the Institute for Molecular and Cell Biology (IBMC), University of Porto, for the assistance in taking the TEM pictures. The authors Ricardo N. Pereira and Oscar L. Ramos also acknowledge FCT for their post-doctoral grants with references SFRH/BPD/81887/2011 and SFRH/BPD/80766/2011, respectively.

Bioprospecting for novel microalgal strains is key to improving the feasibility of microalgae-derived biodiesel production. Tetraselmis sp. CTP4 (Chlorophyta, Chlorodendrophyceae) was isolated using fluorescence activated cell sorting (FACS) in order to screen novel lipid-rich microalgae. CTP4 is a robust, euryhaline strain able to grow in seawater growth medium as well as in non-sterile urban wastewater. Because of its large cell size (9–22 μm), CTP4 settles down after a six-hour sedimentation step. This leads to a medium removal efficiency of 80%, allowing a significant decrease of biomass dewatering costs. Using a two-stage system, a 3-fold increase in lipid content (up to 33% of DW) and a 2-fold enhancement in lipid productivity (up to 52.1 mg L−1 d−1) were observed upon exposure to nutrient depletion for 7 days. The biodiesel synthesized from the lipids of CTP4 contained high levels of oleic acid (25.67% of total fatty acids content) and minor amounts of polyunsaturated fatty acids with ≥4 double bonds (Tetraselmis sp. CTP4 displays promising features as feedstock with lower downstream processing costs for biomass dewatering and biodiesel refining.

Whey is a valuable material with regard to its constitution; however, its disposal is an important problem for the dairy industry, which demands simple and economic solutions. Because lactose is the major component of whey solids, it became interesting and promising to study the bioconversion of lactose components as a substrate for the production of valuable compounds by controlled fermentation processes. The most relevant of these compounds are bioethanol and single cell protein and oil (as the classical ones), organic acids (acetic, lactic, citric, etc.), biogas, biohydrogen, antimicrobial peptides (bacteriocins), enzymes (β-galactosidase and polygalactorunase), bioplastics (polyhydroxyalcanoate and polylactate acid), exopolysaccharides and products obtained from hydrolyzed lactose.

Bovine α-lactalbumin (α-La) and lysozyme (Lys), two globular proteins with highly homologous tertiary structures and opposite isoelectric points, were used to produce bio-based supramolecular structures under various pH values (3, 7 and 11), temperatures (25, 50 and 75 °C) and times (15, 25 and 35 min) of heating. Isothermal titration calorimetry experiments showed protein interactions and demonstrated that structures were obtained from the mixture of α-La/Lys in molar ratio of 0.546. Structures were characterized in terms of morphology by transmission electron microscopy (TEM) and dynamic light scattering (DLS), conformational structure by circular dichroism and intrinsic fluorescence spectroscopy and stability by DLS. Results have shown that protein conformational structure and intermolecular interactions are controlled by the physicochemical conditions applied. The increase of heating temperature led to a significant decrease in size and polydispersity (PDI) of α-La–Lys supramolecular structures, while the increase of heating time, particularly at temperatures above 50 °C, promoted a significant increase in size and PDI. At pH 7 supramolecular structures were obtained at microscale – confirmed by optical microscopy – displaying also a high PDI (i.e. > 0.4). The minimum size and PDI (61 ± 2.3 nm and 0.14 ± 0.03, respectively) were produced at pH 11 for a heating treatment of 75 °C for 15 min, thus suggesting that these conditions could be considered as critical for supramolecular structure formation. Its size and morphology were confirmed by TEM showing a well-defined spherical form. Structures at these conditions showed to be stable at least for 30 or 90 days, when stored at 25 or 4 °C, respectively. Hence, α-La–Lys supramolecular structures showed properties that indicate that they are a promising delivery system for food and pharmaceutical applications., CNPq and CAPES, and their support to FAPEMIG and CNPEM-LNBio (Centro Nacional de Pesquisa em Energia e Materiais-Laboratório Nacional de Biociências) both from Brazil. Fundação para a Ciência e a Tecnologia. The authors thank the FCT Strategic Project of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and the project “BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER.

In recent years, microalgae-based carbon-neutral biofuels (i.e., biodiesel) have gained considerable interest due to high growth rate and higher lipid productivity of microalgae during the whole year, delivering continuous biomass production as compared to vegetable-based feedstocks. Therefore, biodiesel was synthesized from three different microalgal species, namely Tetraselmis sp. (Chlorophyta) and Nannochloropsis oculata and Phaeodactylum tricornutum (Heterokontophyta), and the fuel properties of the biodiesel were analytically determined, unlike most studies which rely on estimates based on the lipid profile of the microalgae. These include density, kinematic viscosity, total and free glycerol, and high heating value (HHV), while cetane number (CN) and cold filter plugging point (CFPP) were estimated based on the fatty acid methyl ester profile of the biodiesel samples instead of the lipid profile of the microalgae. Most biodiesel properties abide by the ASTM D6751 and the EN 14214 specifications, although none of the biodiesel samples met the minimum CN or the maximum content of polyunsaturated fatty acids with a parts per thousand yen4 double bonds as required by the EN 14214 reference value. On the other hand, bomb calorimetric experiments revealed that the heat of combustion of all samples was on the upper limit expected for biodiesel fuels, actually being close to that of petrodiesel. Post-production processing may overcome the aforementioned limitations, enabling the production of biodiesel with high HHV obtained from lipids present in these microalgae.

Using in vitro tests, the efficacy of edible coatings, made using whey protein isolates as a base material and glycerol as a plasticizer, and containing several antimicrobial compounds was assessed upon ubiquitous spoilage microflora in cheese. The formulation that showed the greatest activity was applied onto the surface of experimental cheeses, and their major microbiological and physicochemical features were compared with those of cheese added with commercial coatings. Chitooligosaccharide (COS) and lactic acid exhibited the highest (P< 0.05) bacteriocidal effect against Gram-negative and Gram-positive bacteria, respectively, whereas sodium benzoate and COS exhibited the highest (P< 0.05) inhibitory effect against the yeast. Combinations of lactic acid and COS yielded the highest (P< 0.05) effect against all microorganisms tested; upon application on the cheese surface, they proved to be more active against bacteria, and less effective against yeasts and moulds (P< 0.05) than their commercial coating counterparts.

Consumption of functional probiotic foods has increased over recent decades, alongside with increasing consumer and researcher awareness of their health-promoting effects. This has prompted an interest toward development of novel functional food formulations. Despite fermented dairy products still remaining the most common vectors for the delivery of probiotics to humans, such other food matrices as fruits and vegetables offer a promising performance as sources and carriers of probiotic strains. Hence, these types of matrices are thoroughly reviewed here – with table olives being subjected to comprehensive discussion as a case study, owing to their suitable microstructure and unique sensory and nutritional features.

Broa is an example of bread that is a good candidate for inclusion in functional diets, so it deserves further in-depth study of its chemical composition-namely with regard to evolution of the lipid profile throughout breadmaking, in order to assess whether mixing, fermentation, or baking affect its nutritional value (in terms of unsaturated fatty acids, UFA) based on the assumption that neutral lipids (NL) can be protein- or carbohydrate-bound. Hence, constituent fatty acids in NL of maize (Zea mays) and rye flour (Secale cereale), and in sourdough and final broa manufactured from a mixture therefrom were quantitated. Methodologies of esterification of fatty acids, as well as of transesterification of acyl lipids and sterol esters (SE) were improved. The n-hydrocarbons containing between 4 and 24 carbon atoms were then resolved and identified by gas-liquid chromatography. Regarding total neutral lipids (TNL) in all samples, 79-89% were TAGs, and 87-93% were TAGs and DAGs in the case of free lipids (FL). Furthermore, 73-85% of TNL in bound lipids (BL) and 65-80% of TNL in starch lipids (SL) were TAG and free fatty acids (FFA). Conversely, only 4-5%, 6-16%, and 7-10% of TNL in FL, BL, and SL, respectively, were SE and MAGs. TAGs and DAGs underwent partial hydrolysis during fermentation and baking; palmitic, oleic, and linoleic acids were dominant as products released. The nutritional value of broa lipids was apparent owing to their proportions of SE (4%) and DAG (9%), coupled with 52% of linoleic acid in all samples-as well as to the high contents of polyunsaturated versus monounsaturated or saturated fatty acids, and to the general dominance of UFA.

The contents of free amino acids and biogenic amines in spontaneously fermented sauerkraut, inoculated or not with specific lactic acid bacterium strains, were monitored throughout 45 d of storage. The strains tested were Lactobacillus plantarum 2142, Lactobacillus casei subsp. casei 2763 and Lactobacillus curvatus 2771. In both the control and the experiments, the total amino acid contents increased with time – and the predominant ones were aspartic acid, arginine and glutamic acid. However, upon inoculation with either strain, the total biogenic amine contents remained considerably lower than those of the control (especially in the cases of L. casei subsp. casei and L. curvatus); every single biogenic amine was actually below the 100 ppm-threshold. The dominant biogenic amines in the control were putrescine – and tyramine and histamine, to a lesser extent; the putrescine content was 10-fold lower if inoculation had taken place with either lactobacillus tested; and histamine and tyramine were essentially absent during storage, whereas they ranked above 200 ppm in the control by 45 d. Hence, an efficient food-grade biological tool was made available that constrains buildup of dangerous biogenic amines in fermented vegetables during storage.

A number of enzymes have applications in the textile sector, as several natural fibres (e.g. cotton, wool and flax) can be subjected to processing using such natural biocatalysts. In the latest two decades, demand for industrial enzymes has increased considerably; however, the textile industry requires highly stable enzymes, and good performance at extreme values of pH and temperature. New strategies are continuously emerging for the immobilization of enzymes with superior efficiency and usage. Enzymatic immobilization can stabilize enzymes and extend their useful life. Additionally, reduction in effluent treatment costs and improvement of efficiency is possible. This paper briefly reviews the most recent research efforts pertaining to immobilization of enzymes with potential application in the textile industry.

A total of 13 species of microalgae and 14 strains of cyanobacteria, collected directly in the Portuguese coast and lagoons, were characterized for their fatty acid contents, focusing on two with a market potential — i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); and another already with alternative (yet somehow more expensive) natural sources — i.e. α-linolenic (ALA) acid. The purpose of this work was their eventual inclusion as additives in food or feed. ALA was the most abundant PUFA in Nannochloropsis sp. (0.616 ± 0.081 mgFA.Lculture− 1.d− 1), and EPA in Phaeodactylum tricornutum (0.148 ± 0.013 mgFA.Lculture− 1.d− 1); Pavlova lutheri was particularly rich in EPA (0.290 ± 0.005 mgFA.Lculture− 1.d− 1) and DHA (0.140 ± 0.037 mgFA.Lculture− 1.d− 1). Despite several previous reports on similar topics and encompassing some of our microalgal species, the wild nature of our strains accounts for the novelty of this work — in addition to the characterization of a few wild cyanobacteria. Eustigmatophyceae class was the best producer of ALA, while Prymnesiophyceae was the best for EPA and ALA. Nodularia harveyana exhibited the highest ALA level (0.611 ± 0.022 mgFA.Lculture− 1.d− 1) and Gloeothece sp. was highest in EPA (0.030 ± 0.004 mgFA.Lculture− 1.d− 1).

Reactive forms of oxygen can damage DNA (among other molecules), thus triggering, e.g., atherogenesis and carcinogenesis. However, such dietary antioxidants as lutein and β-carotene can effectively inactivate them; these compounds were found to high levels in a novel strain (M2-1) of the microalga Scenedesmus obliquus. The independent and combined effects of pH and temperature on its rates of growth and production of antioxidants were experimentally assessed, via a full factorial experimental design; the effects of each parameter independently, and of their interactions were accordingly quantified by ANOVA. Our results indicated that temperature plays a more important role on the maximum specific growth rate than pH; in terms of antioxidant content, pH and, to a lesser extent, temperature also have relevant effects. Consequently, the highest rate of biomass specific growth (0.294 ± 0.013 day−1) and biomass productivity (0.837 ± 0.054 mg L−1 day−1) were associated with relatively low pH (6) and relatively high temperature (30°C). Conversely, the antioxidant production rate increased with pH; hence, the highest productivity (0.638 mg L−1 day−1) was attained at pH 8 and 30°C. At the best operating conditions for antioxidant content, the levels of lutein and β-carotene were 203.57 ± 1.41 and 18.20 ± 0.33 mg mL−1, respectively; the maximum production of either one occurred at the early exponential phase. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Aquatic environments are often exposed to toxic heavy metals, which gain access to the food chain via microalgae and may cause severe problems at higher trophic levels. However, such a metabolic specificity can be taken advantage of in bioremediation strategies. The potential of a novel wild strain of Scenedesmus obliquus, previously isolated from a heavy metal-contaminated site in northern Portugal, to remove Zn from aqueous solutions was thus studied, using several initial concentrations. The removal extent reached its maximum by 1 day: 836.5 mg Zn/g biomass, at the initial concentration of 75 mg/L, mainly by adsorption onto the cell surface. Comparative studies encompassing a commercially available strain of the same microalgal species led to a maximum removal extent of only 429.6 mg Zn/g biomass, under identical conditions. Heat-inactivated cells permitted a maximum removal of 209.6 mg Zn/g biomass, at an initial concentration of 50 mg Zn/L. The maximum adsorption capacity of Zn, estimated via Langmuir's isotherm, was 330 mg Zn/g biomass. Finally, Zn removal was highest at pH 6.0-7.0. It was proven, for the first time, that such a wild microalga can uptake and adsorb Zn very efficiently, which unfolds a particularly good potential for bioremediation. Its performance is far better than similar (reference) species, especially near neutrality, and even following heat-treatment.

Bifidobacterium and lactic acid bacteria (LAB), especially from the genera Lactobacillus and Lactococcus, are commonly used in the production of fermented dairy products due to their potential probiotic characteristics. Moreover, some strains of these microorganisms also have the ability to produce conjugated linoleic acid (CLA) from linoleic acid (LA), which has attracted much attention as a novel type of beneficial functional fermented milk. In the present work 22 probiotic bacteria were tested for the production of CLA, using a UV screening method and HPLC techniques. Five microorganisms, two strains of the genera Bifidobacterium, two Lactobacillus and one Lactococcus were selected for their ability to produce CLA after incubation in skim milk with free LA as a substrate. It was possible to quantify the production of CLA (in the range of 40-50μg. CLA/ml) and identify the CLA isomers produced as C18:2 cis 9, trans 11 (60-65%), C18:2 trans 10, cis 12 (30-32%), C18:2 trans 9, trans 11 and C18:2 trans 10, trans 12 (2-5%). © 2010 Elsevier Ltd.

In attempts to optimize their manufacture, whey cheese matrices obtained via thermal processing of whey (leading to protein precipitation) and inoculated with probiotic cultures were tested. A central composite, face-centered design was followed, so a total of 16 experiments were run using fractional addition of bovine milk to feedstock whey, homogenization time, and storage time of whey cheese as processing parameters. Probiotic whey cheese matrices were inoculated with Lactobacillus casei LAFTI®L26 at 10% (v/v), whereas control whey cheese matrices were added with skim milk previously acidified with lactic acid to the same level. All whey cheeses were stored at 7 °C up to 14 d. Chemical and sensory analyses were carried out for all samples, as well as rheological characterization by oscillatory viscometry and textural profiling. As expected, differences were found between control and probiotic matrices: fractional addition of milk and storage time were the factors accounting for the most important effects. Estimation of the best operating parameters was via response surface analysis: milk addition at a rate of 10% to 15% (v/v), and homogenization for 5 min led to the best probiotic whey cheeses in terms of texture and organoleptic properties, whereas the best time for consumption was found to be by 9 d of storage following manufacture.

In Portugal Atlantic codfish (Gadus morhua) is dry-salted with food-grade marine salt for 6 days. During this process, codfish incorporates salt and drains away water up to 22%(w/w) of its weight, which contains important compounds, such as free amino acids, peptides and proteins. Hence, the chemical profile of such water, composed of various soluble nitrogen fractions, was thoroughly determined. Along the salting process, the ratio of water released to trichloroacetic acid-soluble nitrogen remained constant, but the proportion between water released and phosphotungstic acid-soluble nitrogen decreased because of proteolysis. The concentration of free amino acids increased from 2.68 g/l to 5.41 g/l within 6 days, but myofibrillar proteins were found to be approximately constant, ca. 3.31 g/l. Creatine, aspartic and glutamic acids, glycine, taurine and tryptophan were the dominant free amino acids, the release of which was successfully modelled. Total biogenic amines concentration in wastewater at the end of the salting process was ca. 100 mg/kg. (C) 2010 Published by Elsevier Ltd.

A probiotic whey cheese added with Lactobacillus casei LAFTI®L26, Lactobacillus acidophilus LAFTI®L10 or Bifidobacterium animalis Bo was subject in vitro to sequential conditions that parallel the four major steps of digestion: mouth (artificial saliva), oesophagus–stomach (artificial gastric juice), duodenum (artificial intestinal juice) and ileum; its manufacture followed the traditional cheesemaking protocol of Portuguese Requeijao. MRS broth was inoculated in parallel as reference medium, to ascertain the protective effect of the whey cheese matrix itself upon those strains in every digestion step. Mouth conditions had an almost negligible effect upon all three strains, whereas oesophagus–stomach, duodenum and ileum conditions decreased the viable numbers of L. casei and L. acidophilus; in both systems, B. animalis suffered only slight decreases in viable numbers; and L. casei and L. acidophilus behaved likewise in MRS exposed to duodenum and ileum conditions. Whey cheese matrices thus appeared to protect the aforementioned three strains during transit throughout the simulated gastrointestinal system, so they are promising carriers of those probiotic bacteria.

Whey cheeses have been manufactured with probiotic bacteria – viz. Bifidobacterium animalis Bo and Lactobacillus casei LAFTI®L26, from combinations of bovine whey and milk, following protein denaturation at 90 °C; they were subsequently inoculated (at 10%) with those strains, and homogenized afterwards; additives such as salt and sugar were then incorporated; and the resulting solid matrices were stored at 7° C for up to 21 d. Oscillatory measurements and instrumental texture profile analyses were performed, and sensory analyses were carried out by a trained panel. Microstructural features were in addition ascertained by scanning electron microscopy. L. casei exhibited a higher acidifying activity than B. animalis, which produced distinct textures; higher firmness and viscoelasticity were indeed found in matrices inoculated with the former. Incorporation of sugar and L. casei favoured consumer acceptability, relative to plain matrices. Microstructural differences were detected between matrices at different times of storage and formulated with distinct additives.

This research effort was aimed at assessing the changes in extractable neutral lipids (NL) throughout the baking process of Broa, a Portuguese traditional sourdough bread. NL were accordingly isolated, purified and quantitated - starting from non-starch lipid (NSL) and starch lipid (SL) extracts of maize and rye flours, as well as fermented dough and bread. NSL accounted for the major fraction of extracted lipids; furthermore, the NSL/SL ratio evolved throughout processing in agreement with the phenomena prevailing during dough preparation, fermentation, and baking. An analytical method based on resolution by normal-phase HPLC coupled with detection by evaporative light scattering was accordingly developed for quantitation of the aforementioned NL classes. Distinct NL classes correlated well with the stage of bread making. The main NL in NSL were triacylglycerols (ca. 75% of the total), but relatively high concentrations of sterol esters and diacylglycerols were also found. Conversely, free fatty acids were the dominant component of SL, whereas monoacylglycerols and free sterols were comparable to those in NSL.

The microbiological profile in raw milk cheeses is typically characterized by a multitude of microbial groups, with interactions among them throughout ripening that are not fully understood to date. Incidence of undesired microorganisms in raw cheesemaking milk, as is the case of either spoilage or even pathogenic ones, is a common trait in Portuguese traditional cheeses. Hence, they will likely contribute to the physicochemical changes occurring therein and, consequently, to the characteristics of the final product. In order to gain insight into their role, model cheese systems, manufactured as far as possible according to artisanal practices (except that the initial microbial load and biodiversity were controlled), were experimentally tested. Single contaminants, or a consortium thereof, were inoculated at two levels in sterilized raw ewe's milk, and duly combined with inocula containing one or two lactic acid bacteria normally found in those traditional cheeses. The physicochemical composition, organic acid profile, and evolution of both protein breakdown and rheology were monitored throughout a 60 d-ripening period. Modifications brought about within the cheese matrix as a result of microbial metabolism, especially those arising from the interaction between lactic acid bacteria and unwanted microorganisms, included the enhanced release of peptides and free amino acids, which in turn led to higher viscoelastic moduli. The final model cheeses could be well discriminated, based on the impact of the various inocula considered upon the levels of organic acids. Conversely, proteolysis and viscoelastic properties appeared to be essentially independent of the initial microflora.

Aquaculture is a growing commercial activity worldwide, which resorts more and more often to microalgae as feed; the lipid composition of such microalgae is a critical factor with regard to the fish growth rate upon ingestion. The aim of this work was thus to study the influence of light intensity on the lipid profile of a known microalga, Pavlova lutheri. Several semi-continuous cultures were carried out, and biochemical parameters such as lipid, protein, carbohydrate, and chlorophyll contents were quantified. Lipids were specifically fractionated into classes by TLC, and those in each class were subjected to GC afterwards in an attempt to ascertain their fatty acid profile. Evidence was consequently provided which showed that cultures grown under low light intensity (9 W m−2) possess a higher fraction of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids esterified in polar classes—which are those with a more favorable role in aquaculture. It was also demonstrated that intermediate levels of light intensity (19 W m−2) may be misleading in terms of favorable effects upon EPA and DHA contents—because there is an increase in their total yields and productivities, but they appear mostly esterified into triacylglycerols; this may be a favorable deed for production and purification, but is metabolically not so effective in aquaculture. The highest EPA and DHA productivities attained were 1.29 and 0.69 mg L−1 day−1, respectively, at intermediate levels of light intensity (19 W m−2).

Respiration rates were measured in fresh-cut ‘Rocha’ pear (Pyrus communis L.) stored at four temperatures (0, 5, 10 and 15 °C) and with oxygen partial pressures ranging from 0 to 18 kPa. Respiratory quotient and ethanol production were used to determine the fermentation threshold. The oxygen concentration effect on the respiration rate was accurately described using Michaelis–Menten kinetics, without non-competitive inhibition by CO2, and the effect of temperature on the respiration rate was well modelled by exponential functions. The oxygen level at which respiration was half its maximum (apparent K m,O 2 ) was similar to or only slightly greater than the fermentation threshold. The narrow range of oxygen between K m,O 2 and the fermentation threshold, suggests that modified atmosphere packaging technology has a limited applicability toward extension of the shelf-life of fresh-cut ‘Rocha’ pear.

The dependence of the extent of aqueous extraction of antioxidant compounds on particle size and contact time was studied for three important medicinal plants, that are commonly used in infusions: agrimony, sage and savoury. The effect of extraction time was dependent on the plant considered; however, ca. 5 min can be taken as the minimum period required to assure an acceptable degree of extraction of those compounds. As expected, a smaller particle size led to a higher extraction extents; a typical value of 0.2 mm is accordingly recommended. Chlorogenic acid was the dominant phenolic compound extracted from agrimony, whereas caffeic acid dominated in the case of sage or savoury. A mathematical model based on Fick’s law was developed from first principles, and its two parameters were suitably fitted to the experimental data generated – in attempts to predict the evolution of antioxidant capacity extracted during contact time, for each plant and each particle size.

The behavior of Bifidobacterium animalis subsp. lactis Bb 12 under batch cultivation, after continuous culturing for up to 12 d, was monitored in skim milk-based media. Previous continuous culture for longer than 6 d affected the physiology of said microorganism. The minimum inhibitory concentrations of lactic and acetic acids increased from 18 to 26 g/l, whereas the molar ratio of acetic to lactic acid increased from 0.8 to 1.55, when the previous continuous culture increased its duration from 1 to 12 d. The specific lactose consumption rate decreased from 0.94 to 0.77 glactose/gcell dry mass/h within the batch culture timeframe; this was concomitant with greater amounts of acetic and formic acids, and lower amounts of lactic acid produced. The β-galactosidase activity increased as continuous culturing time increased, and reached 446 units/ml by 12 d; however, the rate of enzyme synthesis decreased concomitantly. Succinic acid was produced during the exponential growth and stationary phases of the batch culture, but the former at exponential growth phase was higher as the continuous culturing time was longer. For comparison purposes, batch cultivation of samples taken from continuous cultures by 1 and 12 d was done using a semi-synthetic medium with glucose as carbon source; a pattern similar to that observed when using skim milk-based media was observed.

Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely distributed in nature. Its intrinsic thermal resistance coupled with the extraordinary resistance against common food preservation techniques makes it one of the most frequent food-poisoning microorganisms causing both intoxications and infections. In order to control B. cereus growth/sporulation, and hence minimize the aforementioned hazards, several antimicrobial compounds have been tested. The aim of this work was to assess by atomic force microscopy (AFM) the relationship between the molecular weight (MW) of chitosan and its antimicrobial activity upon both vegetative and resistance forms of B. cereus. The use of AFM imaging studies helped us to understand how chitosans with different MW act differently upon B. cereus. Higher MW chitosans (628 and 100 kDa) surrounded both forms of B. cereus cells by forming a polymer layer-which eventually led to the death of the vegetative form by preventing the uptake of nutrients yet did not affect the spores since these can survive for extended periods without nutrients. Chitooligosaccharides (COS) (

Bacteriophage/bacterium systems have been employed in the past in assays for virucidal activity. A novel application of one such system is proposed here for the in vivo determination of antioxidant capacity. It was shown that an antioxidant such as gallic acid can effectively protect against oxidative damage brought about by H2O2-but only within a narrow range of concentrations (i.e., from 250 to 500 mg L-1); ascorbic acid, on the other hand, did not exhibit any protective effect against H2O 2. Finally, neither ascorbic nor gallic acid demonstrated a virucidal effect. The P22/Salmonella typhimurium model system thus proved to be useful in the assessment of antioxidant capacity in vivo, at least using those two alternative model antioxidants.

The aim of this research effort was to investigate the role of various sugar substrates in the growth medium upon thermotolerance and upon survival during storage after freeze-drying of Lactobacillus bulgaricus. Addition of the sugars tested to the growth medium, and of these and sorbitol to the drying medium (skim milk) was investigated so as to determine whether a relationship exists between growth and drying media, in terms of protection of freeze-dried cells throughout storage. The lowest decrease in viability of L. bulgaricus cells after freeze-drying was obtained when that organism was grown in the presence of mannose. However, L. bulgaricus clearly survived better during storage when cells had been grown in the presence of fructose, lactose or mannose rather than glucose (the standard sugar in the growth medium). A similar effect could not be observed in terms of thermotolerance; in this case, the growth medium supplemented with lactose was found to yield cells bearing the highest heat resistance. Supplementation of the drying medium with glucose, fructose, lactose, mannose or sorbitol led in most cases to enhancement of protection during storage, to a degree that was growth medium-dependent.

BACKGROUND: Aqueous extracts of 48 herbal plants were obtained via alternative extraction protocols, and were assayed for their capacity to protect deoxyribose and DNA itself from degradation (or, conversely, for their capacity to promote DNA degradation), using electrophoresis as analytical tool. RESULTS: For a given (constant) volume of extract, deoxyribose protection ranged from 14.13 ± 1.35% (mean ± SD) inhibition by dwarf mallow powder infusion, up to 106.51 ± 15.93% inhibition by avocado powder infusion. DNA protection was tested at two extract concentrations, and was slightly greater at the higher concentration. Pro-oxidant effects were essentially absent. CONCLUSION: The anti-oxidative roles of plants upon deoxyribose and DNA displayed by our experimental results were rather promising with regards to practical applications of those plants, viz. as ingredients in the formulation of nutraceutical beverages and/or foods. Copyright © 2007 Society of Chemical Industry

Summary Under the trade name lipolysed milkfat (LMF), concentrated cheese flavours obtained via enzymatic treatments of cheese (or other dairy substrates) have been made available in increasing numbers. In this research effort, (anhydrous) milkfats from cows, ewes and goats were used as substrates for production of cheesy flavours. Those milkfats were subjected to modifications brought about by ten different (commercial) lipases and one cutinase, and the free fatty acids released were analysed by high performance liquid chromatography. Both the degrees of hydrolysis and the free fatty acid profiles of the final products were similar to those reported for several LMF products, although specific keynotes could be pinpointed. Consequently, those milkfats may represent alternative raw materials for manufacture of cheesy flavours, while contributing to alleviate the problem derived from the increasing surplus of milkfat in Western countries.

BACKGROUND: Aqueous extracts of most medicinal plants traditionally employed in Portugal (at the ratio of 1 g plant: 110 mL water) have been assayed for total antioxidant capacity and phenol content, in order to elucidate their claimed medicinal features. RESULTS: The antioxidant activity was assessed by the ABTS•+ method; the ascorbic acid equivalent values ranged from 1.4280 ± 0.1261 g L-1 for avocado (Persea americana (Lauraceae)) obtained by infusion of powder, down to 0.0027 ± 0.0012 g L-1 for olive (Olea europaea (Oleaceae)) obtained by infusion of leaves. Total phenol content was determined by the Folin-Ciocalteu procedure; the gallic acid equivalent values ranged from 0.5541 ± 0.0289 g L -1 for avocado obtained by infusion of powder, down to 0.0053 ± 0.0014 g L-1 for olive obtained by boiling leaves. A good correlation between total antioxidant capacity and total phenol content was found. CONCLUSION: The method of powder infusion should be chosen if high concentration of antioxidants are sought. On the other hand, a high antioxidant capacity and a high phenol content correlate well with the empirically established (and widely publicised) capacity to treat respiratory infections.

The objective of this research work was to investigate the antioxidant properties of sterilized yacon tuber flour. The results revealed for the first time the high antioxidant activity of sterilized yacon flour. The best extract obtained by boiling 8.9% (w/v) of yacon flour in deionised water for 10 min exhibited a total antioxidant capacity of 222 ± 2 mg (ascorbic acid equivalent)/100 g DW and a total polyphenol content of 275 ± 3 mg (gallic acid equivalent)/100 g DW associated to the presence of four main phenolic compounds: chlorogenic acid, caffeic acid, coumaric acid and protocatechuic acid, as well as the amino acid tryptophan. The most abundant was chlorogenic acid, followed by caffeic acid. Biological assays revealed that the extract had indeed antioxidant protection, and no pro-oxidant activity. In conclusion, sterilized yacon tuber flour has the potential to be used in the food industry as a food ingredient to produce functional food products.