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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

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.

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.

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

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.

Spontaneous fermentation constitutes the basis of the chief natural method of processing of table olives, where autochthonous strains of lactic acid bacteria (LAB) play a dominant role. A thorough literature search has unfolded 197 reports worldwide, published in the last two decades, that indicate an increasing interest in table olive-borne LAB, especially in Mediterranean countries. This review attempted to extract extra information from such a large body of work, namely, in terms of correlations between LAB strains isolated, manufacture processes, olive types, and geographical regions. Spain produces mostly green olives by Spanish-style treatment, whereas Italy and Greece produce mainly green and black olives, respectively, by both natural and Spanish-style. More than 40 species belonging to nine genera of LAB have been described; the genus most often cited is Lactobacillus, with L. plantarum and L. pentosus as most frequent species-irrespective of country, processing method, or olive type. Certain LAB species are typically associated with cultivar, e.g., Lactobacillus parafarraginis with Spanish Manzanilla, or L. paraplantarum with Greek Kalamata and Conservolea, Portuguese Galega, and Italian Tonda di Cagliari. Despite the potential of native LAB to serve as starter cultures, extensive research and development efforts are still needed before this becomes a commercial reality in table olive fermentation. info:eu-repo/semantics/publishedVersion

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.

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.

Microalgal biomass can be “energy rich,” but the growth of algae in dilute suspension around 0.02%–0.05% dry solids is a considerable challenge in achieving a viable energy balance in a microalgal biofuel process. Many researchers consider that efficient harvesting is the major challenge of microalgal biofuel commercialization but a single method or combination of harvesting methods suited to all microalgae does not exist. Additionally, lipid extraction continues to be a significant trial even though a multitude of extraction methods, especially in what concerns the presence of residual water in the microalgal biomass; its effects on lipid extraction or the need of cell disruption are still not well understood. In this chapter, these critical points on biofuel production—biomass harvesting and dewatering, biomass pretreatment, and fatty acid extraction methods—will be discussed.

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

Antioxidants are secondary metabolites in plants, designed to protect them from abiotic stress; however, they may also improve one's general health, following regular ingestion. Since most foods from plant origin are consumed only after processing and formulation, the final activity exhibited by their antioxidants may be rather different from that in the original plant. Ten plants empirically used in Portugal in traditional medicine were accordingly studied - agrimony (Agrimonia eupatoria), eucalyptus (Eucalyptus globulus), walnut-tree (Juglans regia), myrtle (Myrtus communis), raspberry (Rubus idaeus), sage (Salvia sp.), savory (Satureja montana), sweet-amber (Hypericum androsaemum), thyme (Thymus vulgaris) and yarrow (Achillea millefolium), for total antioxidant capacity and total phenolic content. Significant variations were found between fresh and frozen forms: most plants decreased those features by 30-80%. However, weather conditions prevailing during plant growth also had a significant impact, besides postharvest storage conditions especially in the case of antioxidant capacity. Typically, a decrease occurred throughout processing and storage, which was maximum for myrtle and minimum for yarrow. The results of this research are useful in attempts to preserve the antioxidant content of plant-derived foods, or of plant additives in foods, via rational manipulation of processing conditions after harvest and throughout storage. (C) 2012 Elsevier Ltd. All rights reserved.

The main purpose of this study was to isolate and briefly characterize proteolytic bacteria from a poorly known habitat - raw wool. Fleece samples were accordingly collected from Merino raw wool - a Portuguese ewe breed, at three distinct areas of their body, from animals exhibiting no symptoms or signs of abnormalities: they were then subjected to enumeration and isolation of a total of 158 bacterial strains. Said isolates were screened for protease activity, using the spot technique, on Calcium Caseinate Agar containing 1% (w/v) skim milk. The 36 isolates displaying the highest protease activity underwent a more refined assessment of enzymatic performance - by examining their cell-free supernatant extracts, using casein as substrate. Two Bacillus isolates were eventually chosen owing to their highest proteolytic activities (24.6 and 15.9 U/mL), and identified using molecular biology tools. (C) 2012 Elsevier B.V. All rights reserved.

The aim of this study was to investigate the stability of Enterococcus strains isolated from a traditional Portuguese cheese and previously proved to be safe, in dairy matrices, and to assess survival of the best strains in the human gastrointestinal (GI) tract. Enterococcus faecium 32 and Enterococcus durans 37 were added to yoghurt that was ingested by 4 healthy adults. Detection of the enterococcal strains was performed with RAPD-PCR. The intervention trial showed transient colonisation with both strains, via presence in faeces during the ingestion period and disappearance by 10 d post-ingestion. Viable numbers of enterococci increased during the consumption period by 1.8–4.4 log-values, and returned to baseline level during the follow-up period. Based on data of the dairy matrix stability trials and human intervention study involving yoghurt ingestion, E. faecium 32 survived well both in the food matrix and in the human GI tract, thus showing probiotic potential.

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.

A thorough microbiological study of maize and rye flours, and sourdoughs obtained therefrom for eventual manufacture of broa – a dark sour bread typical in Northern Portugal, following artisanal practices, was carried out. Towards this purpose, samples were supplied by 14 artisanal producers, selected from 4 sub-regions, during two periods of the year. Total viable counts, as well as viable mesophilic and thermophilic microorganisms, yeasts and molds, Gram− rods, endospore-forming and nonsporing Gram+ rods, and catalase+ and catalase− Gram+ cocci were assayed for. The comprehensive experimental dataset unfolded a unique and rather complex wild microflora in flours and sourdoughs throughout the whole region, which did not discriminate among sub-regions or seasons, or flour source for that matter. However, fermentation played a major role upon the numbers of the various microbial groups: the viable counts of yeasts, lactobacilli, streptococci, lactococci, enterococci and leuconostocs increased, whereas those of molds, Enterobacteriaceae, Pseudomonadaceae, staphylococci and micrococci decreased.

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.

Molecular hydrogen (H 2 ) obtained from biological sources provides an alternative to bulk chemical processes that is moving towards large-scale, economical generation of clean fuel for automotive engines. This opinion article examines recent improvements in H 2 production by wild and mutant strains of Chlamydomonas reinhardtii – the green microalga currently considered the best eukaryotic H 2 producer. Here, we review various aspects of genetic and metabolic engineering of C. reinhardtii , as well as of process engineering. Additionally, we lay out possible scenarios that would lead to more efficient research approaches in the near future, as part of a consistent strategy for sustainable biohydrogen supply.

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).

Carbon-neutral renewable liquid biofuels are needed to displace petroleum-derived transport fuels in the near future - which contribute to global warming and are of a limited availability. A promising alternative is conveyed by microalgae, the oil content of which may exceed 80% (w/wDW) - as compared with 5% of the best agricultural oil crops. However, current implementation of microalga-based systems has been economically constrained by their still poor volumetric efficiencies - which lead to excessively high costs, as compared with petrofuel prices. Technological improvements of such processes are thus critical - and this will require a multiple approach, both on the biocatalyst and bioreactor levels. Several bottlenecks indeed exist at present that preclude the full industrial exploitation of microalgal cells: the number of species that have been subjected to successful genetic transformation is scarce, which hampers a global understanding (and thus a rational design) of novel blue-biotechnological processes; the mechanisms that control regulation of gene expression are not fully elucidated, as required before effective bioprocesses based on microalgae can be scaled-up; and new molecular biology tools are needed to standardize genetic modifications in microalgae - including efficient nuclear transformation, availability of promoter or selectable marker genes, and stable expression of transgenes. On the other hand, a number of pending technological issues are also present: the relatively low microalga intrinsic lipid productivity; the maximum cell concentration attainable; the efficiency of harvest and sequential recovery of bulk lipids; and the possibility of by-product upgrade. This review briefly covers the state of the art regarding microalgae toward production of biofuels, both from the point of view of the microalgal cell itself and of the supporting bioreactor; and discusses, in a critical manner, current limitations and promising perspectives in this field.

The functional properties of the dominant adventitious yeast strains in Portuguese cultivars of brined olives were evaluated. Identification followed traditional taxonomic methods, complemented with molecular biology approaches. The yeast population ranged in 3–5 log10 (cfu/mL), and included chiefly Pichia membranaefaciens, Pichia fermentans, Saccharomyces cerevisiae and Candida oleophila. A few strains exhibited desirable technological features, viz. absence of pectinolytic and lipolytic activities, positive catalase response, high osmotolerance, ability to uptake oleuropein and lactic acid, and capacity to produce B-complex vitamins. Furthermore, antimicrobial activity against selected food-borne bacterial pathogens was observed, as well as release of mycocin. P. membranaefaciens and C. oleophila appeared as the most promising candidates for eventual inclusion in tailor-made probiotic starter/adjunct cultures.

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.

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.

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.

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.

The aim of this study was to assess the hygienic quality of raw milk used in the manufacture of São Jorge, a Protected Denomination of Origin Portuguese semihard cheese, as well as to ascertain the sanitary conditions prevailing during its processing. Viable counts of Enterobacteriaceae and Micrococcaceae were accordingly obtained, pertaining to 21 independent batches (including samples of raw milk, curd, and cheeses after 1, 3, and 4 months of ripening), from 7 dairy farms. Standard plate counts (log CFU per milliliter or per gram) ranged from 6.1 to 8.6 in raw milk, whereas they ranged from 7.0 to 8.0 in 4-month-old cheeses. Viable counts of Enterobacteriaceae ranged between 5.9 and 7.0 in raw milk and between 0.0 and 1.3 in 4-month-old cheeses. Species identified within this family encompassed Klebsiella oxytoca, Klebsiella cloacae, Klebsiella pneumoniae, Enterobacter sakazakii, and Escherichia coli; Klebsiella ornithinolytica, Klebsiella terrigena, and Serratia odorifera were detected only in raw milk. No Salmonella whatsoever could be detected in any of the samples. Viable counts of Micrococcaceae ranged between 4.7 and 5.9 and between 1.3 and 3.3 in raw milk and 4-month-old cheeses, respectively. Species identified within this family encompassed Staphylococcus sciuri, Staphylococcus epidermidis, Staphylococcus saprophyticus (which was found mainly in ripened cheeses), and Staphylococcus aureus (which was not detected in 4-month-old cheeses). Accompanying physicochemical analyses included determination of moisture, salt, and pH. Statistical analyses revealed a negative correlation between salt content and viable numbers of Enterobacteriaceae in cheese, whereas in the case of Micrococcaceae, a more negative correlation was found between viable numbers and moisture content than between viable numbers and pH. The results of our study indicate, in general, poor milk handling conditions in all farms, given that the indicators total mesophile and Enterobacteriaceae counts were high, between 100- and 1,000-fold those enforced by international standards pertaining to the matrices in question. However, by the time of regular consumption (i.e., after 4 months of ripening), São Jorge cheeses exhibit low levels of contamination by Enterobacteriaceae and S. aureus, as well as absence of Salmonella.

Enterococci account for an important fraction of the adventitious microflora of traditional cheeses manufactured in Mediterranean countries from small ruminants' raw milk and play an important role in the development of suitable organoleptic characteristics of the final product. It has been suggested that animals used for food or animals that supply edible products are a reservoir of antibiotic-resistant enterococci. The main purpose of this research effort was thus to identify, to the species level, a total of 73 enterococci with high tolerance to acidic pH and bile salts (as prevailing environmental conditions in the first portion of the gastrointestinal tract), which were previously isolated from the milk feedstock to the final product of Terrincho cheesemaking, and to determine their profiles of antibiotic susceptibility, coupled with the occurrence of specific virulence factors (especially in those that might eventually be claimed to exhibit suitable probiotic and technological performances). Isolates, identified by both API 20 STREP and PCR methods, were found to belong to the following Enterococcus species: E. casseliflavus, E. durans, E. faecalis, E. faecium, and E. gallinarum. Susceptibility of those isolates was observed to most antibiotics tested, whereas none harbored aminoglycoside resistance genes. PCR screenings for cytolysin genes (cylL(L), cylL(s), cylM, cylB, and cylA), surface adhesin genes (efaA(fs), efaA(fm), and esp), the aggregation protein gene (agg), and the extracellular metalloendopeptidase gene (gelE) were performed. All isolates proved negative for cylL(L), cylM, cylB, and agg genes. Both E. faecalis strains were positive for the cell wall-associated protein Esp and the cell wall adhesin efaA(fs), whereas the cell wall adhesin efaA(fm) was detected in 11 of the 12 E. faecium strains. Only one strain possessed the cylL(s) determinant, and another possessed the cylA gene. Incidence of virulence determinants was thus very low; hence, the enterococcal adventitious microflora tested is essentially safe.

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.

This work presents the first study on the bacterial communities in Pico cheese, a traditional cheese of the Azores (Portugal), made from raw cow's milk. Pyrosequencing of tagged amplicons of the V3–V4 regions of the 16S rDNA and Operational Taxonomic Unit-based (OTU-based) analysis were applied to obtain an overall idea of the microbiota in Pico cheese and to elucidate possible differences between cheese-makers (A, B and C) and maturation times. Pyrosequencing revealed a high bacterial diversity in Pico cheese. Four phyla (Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes) and 54 genera were identified. The predominant genus was Lactococcus (77% of the sequences). Sequences belonging to major cheese-borne pathogens were not found. Staphylococcus accounted for 0.5% of the sequences. Significant differences in bacterial community composition were observed between cheese-maker B and the other two units that participated in the study. However, OTU analysis identified a set of taxa (Lactococcus, Streptococcus, Acinetobacter, Enterococcus, Lactobacillus, Staphylococcus, Rothia, Pantoea and unclassified genera belonging to the Enterobacteriaceae family) that would represent the core components of artisanal Pico cheese microbiota. A diverse bacterial community was present at early maturation, with an increase in the number of phylotypes up to 2 weeks, followed by a decrease at the end of ripening. The most remarkable trend in abundance patterns throughout ripening was an increase in the number of sequences belonging to the Lactobacillus genus, with a concomitant decrease in Acinetobacter, and Stenotrophomonas. Microbial rank abundance curves showed that Pico cheese's bacterial communities are characterized by a few dominant taxa and many low-abundance, highly diverse taxa that integrate the so-called “rare biosphere”.

Yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Robinson; Asteraceae] roots have been shown to be a source of prebiotic compounds. However, there are no known studies concerning processed yacon roots. The objective of this study was to investigate the potential prebiotic activity of yacon tuber flour. For this purpose, an aqueous extract was tested for selection of yacon incorporation and sterilization method and selection of the most favourable concentration to be tested for prebiotic activity. Once these conditions were identified, the potential prebiotic activity of the yacon extract was evaluated by determination of viable cell numbers and metabolic activity against four probiotic strains, namely, Enterococcus faecium 32, Bifidobacterium animalis Bo, Lactobacillus acidophilus Ki and Lactobacillus casei L26). Results showed that the best incorporation and sterilization method was to autoclave the supernatant, resultant from the yacon tuber flour suspension, at 121 degrees C for 20 min and add it to sterilized basal medium. For the confirmation of potential prebiotic activity, de Man-Rogosa-Sharpe (MRS) medium without a conventional carbon source (negative control), with 2% (w/v) glucose per se (positive control) and associated with 1% (w/v) yacon tuber flour were chosen. Yacon tuber flour revealed a potential prebiotic activity upon the growth of the probiotic strains tested, probably due to its fructooligosaccharides (FOS) content. (C) 2015 The Institution of Chemical Engineers.

The research effort described here has focused on incorporation of Lactobacillus casei , in whey protein matrices, in the presence of selected salty additives. Those matrices were produced via thermal processing of a combination of either ovine or bovine whey (or a mixture thereof) with ovine milk, and were inoculated (at 10%) with L. casei strain LAFTI ® L26; salt, salt and herbs, or salt and xanthan were further added to such matrices, which were then homogenized and stored at 7 °C for up to 21 d. In general, viable cell numbers maintained or even increased throughout the storage period, irrespective of the type of salty additive considered. Partial depletion of lactose was detected, and concomitant production of lactic acid throughout the 21 d-period of storage; lower lactic acid concentrations were found in matrices containing salty additives. In matrices with xanthan (SX), the probiotic strain exhibited the lowest metabolic activity. Matrices SX were less soft and firmer than the others, by the end of storage, and were similar to matrices with herbs (SH). The incorporation of salty additives affected bacterial metabolism, in terms of glycolysis and proteolysis, which in turn had a significant impact on the development of textural properties.

Use of probiotic bacteria and consumes in large — in novel foods to provide beneficial health effects has attracted an increasing interest by the food industry and fermented olives are an excellent example of a new generation of those foods from plant origin so as to assure maximum viability by the time of ingestion during processing and storage of food products, as well as during transit through the gastrointestinal tract. Our study focused on production, characterization and assessment of efficacy of microencapsulation upon survival of probiotic strains and sensory properties of the final olive paste throughout refrigerated storage. Microencapsulation appears to be an effective technique for strain survival, depending on the operating temperature and experimental results on tolerance to gastrointestinal-like conditions, and ability to adhere to intestinal epithelium is thereby presented and discussed. The sensory panel rated all experienced matrices as good, including overall acceptance without significant preference between them. However, the success of microencapsulation was more limited when incorporated into olive paste. Free cells of Lactobacillus plantarum 33 proved able to survive in olive paste during storage at refrigerated temperatures.

The contributions of the coagulant Cynara cardunculus and of the microflora of raw milk to the volatile-free fatty acid profile of Serra da Estrela cheese were evaluated. The experimental design included both a model system and, dual cheeses. The study in the model system showed that isovaleric acid was the predominant volatile compound after 7 d of ripening. The systems inoculated with Enterococcus faecium produced the highest amount of this volatile (ca. 135.8 mg kg-1 curd), while those inoculated with Lactobacillus plantarum produced the least (21.4 mg kg-1 curd); Lactococcus lactis produced moderate amounts (ca. 34.2 mg kg-1 curd) but a total amount of volatile-free fatty acids similar to those found in control samples. This is considered advantageous since this volatile fatty acid confers a harsh, piquant, mature flavour to cheese, coupled with the realisation that excess volatiles may result in off-flavours. The addition of cultures in experimental cheeses helped reduce ripening time to about one half. Inclusion of Lb. plantarum led to cheeses containing the highest amounts of volatiles, and exhibiting an aroma closest to that of typical Serra da Estrela cheese.

The viabilities of probiotic strains of ains of Lactobacillus acidophilus, Lactobacillus paracasei, Bifidobacterium animalis, and Lactobacillus brevis were studied following incorporation in a whey cheese matrix. Experimental were studied following incorporation in a whey cheese matrix. Experimental production of plain, as well as sugar-added or salt-added whey cheeses, was based on the traditional manufacture protocol of Requeijão, a Portuguese whey cheese that essentially results from protein denaturation via heating of whey at about 85 °C. After inoculation, the experimental whey cheeses were incubated at 7 °C for 28 d. Our results have shown that all strains considered were able to maintain (or even increase) their initial viable numbers; have shown that all strains considered were able to maintain (or even increase) their initial viable numbers; L. paracasei paracasei ssp. paracasei paracasei strain LCS-1 and strain LCS-1 and L. acidophilus L. acidophilus strain Ki exhibited the highest cell viability in plain Requeijão by the end of the storage period—an increase of ca. 2 log cycles in their viable numbers was actually recorded. by the end of the storage period—an increase of ca. 2 log cycles in their viable numbers was actually recorded. Among the parameters studied, bacterial species and matrix nature had the most important effect upon viable counts, whereas time of storage was the least important. counts, whereas time of storage was the least important.

From the extracellular fluid of a novel strain of Bjerkandera sp., it was isolated, purified and identified the main enzyme responsible for Remazol Brilliant Blue R dye decolourisation. Such an enzyme is able to oxidise manganese, as well as veratryl alcohol and 2,6-dimethoxyphenol in manganese-independent reactions; hence, it can be included in the new group of versatile peroxidases. The molecular mass of said enzyme is ca. 45 kDa, and the N-terminal amino acid sequence obtained by Edman degradation is VAXPDGVNTA. The enzyme substrate range for oxidation of several phenolic and non-phenolic aromatic compounds was determined and the corresponding Michaelis–Menten kinetic constants calculated. Furthermore, spectrophotometric assays showing the Soret band and allowing observation of band shifts of the enzyme led to the conclusion that Bjerkandera strains may also synthesise at least two different versatile peroxidases, as happens with Pleurotus eryngii .

Studies encompassing variation of milk clotting time with the concentration of proteases extracted from Cynara cardunculus were performed; milk clotting time dependence is not linear, and a model was postulated that fits well the experimental data obtained, and hence may be useful in predicting changes during the cheesemaking process. Parallel studies were also conducted pertaining to proteolysis of a mixture of ovine and caprine caseins, in attempts to investigate the stability of the aforementioned coagulating enzymes in crude or in pure form, with or without previous incubation for a certain time under typical ripening conditions. The enzymes exhibited an increase in activity whenever previous incubation had taken place. Moreover, the extent of enzyme-mediated proteolysis was always higher on caprine than on ovine milk caseins.

The enzymatic hydrolysis of the major whey proteins, namely β-lactoglobulin (β-Lg) and α-lactalbumin (α-La), was experimentally studied using whey as substrate; an aspartic protease (cardosin A), previously extracted from the flowers of Cynara cardunculus and purified by gel filtration and ion exchange chromatographies, was used for this purpose. Sweet whey was incubated for 24 h at various enzyme:substrate ratios, at controlled pH (5.2 and 6.0) and temperature (55 °C); the hydrolyzates were assayed by gel permeation chromatography and electrophoresis. A mechanistic model was proposed for the kinetics, which basically leads to a double-substrate, single-enzyme Michaelis–Menten rate expression containing four adjustable parameters; these parameters were estimated by applying multiresponse, nonlinear regression analysis to the experimental data, so that the model would yield good fits. The best estimates obtained for Km were markedly lower for α-La than for β-Lg, so cardosin A shows a higher affinity for α-La than for β-Lg. The experimental results also suggest that β-Lg is rather resistant to enzyme-mediated hydrolysis under all experimental conditions tested. The highest activity (measured by kcat) of cardosin A was recorded toward α-La (i.e. 0.013 s−1) at pH 5.2. Furthermore, the specificity ratio (kcat/Km), obtained toward each whey protein, indicated that cardosin A possesses a higher catalytic efficiency for hydrolysis of α-La than of β-Lg; the highest value for this ratio was recorded for α-La at pH 5.2, and was close to that reported elsewhere for cardosin A acting on caseins and casein-like substrates.

The industrial exploitation of lactic acid bacteria (LAB) as starter and/or probiotic cultures depends strongly on the preservation technologies employed, which are required to guarantee long-term delivery of stable cultures in terms of viability and activity. Freeze-dried preparations exhibit advantages relative to preparations made with other techniques in terms of long-term preservation, coupled with convenience in handling, storage, marketing and application. Degrees of survival of LAB cultures as high as possible, during drying and subsequent storage, are thus of nuclear importance, both technologically and economically. This review covers several factors, deemed relevant for preservation of freeze-dried LAB. The state of the art of the knowledge focussed on the improvement of LAB survival during freeze-drying and subsequent storage is presented, including specific discussion of the effects of (i) intrinsic factors, (ii) growth factors, (iii) sub-lethal treatments, (iv) drying media and (v) storage and rehydration.

Cheesemaking from batches of raw ewe’s milk was carried out via inoculation with wild strains of Lactococcus lactis subsp. lactis ESB110019 and Lactobacillus plantarum ESB5004 independently, or combined with each other. Those two strains had been isolated from the native microflora of typical Serra da Estrela cheese. One control batch was processed in parallel without addition of any starter. The evolution in viable counts of the main micro-organisms (viz. lactic acid bacteria, Enterobacteriaceae, staphylococci and yeasts), as well as in secondary proteolysis (WSN, 2% TCASN, 12% TCASN and 5% PTASN), was monitored throughout ripening time (over a 63-day period) in cheeses from each batch. The sensory features of the fully ripened cheeses were also assessed. Cheeses manufactured with starter showed significantly lower levels of viable Enterobacteriaceae than those manufactured without starter; viable counts of enterococci and staphylococci did significantly increase after addition of L. lactis or Lb. plantarum, respectively. Proteolysis in terms of WSN and 5% PTASN was not significantly affected by the lactic acid bacteria tested when compared to the control, but L. lactis played a significant role toward increasing the 2% TCASN content of cheeses; both strains led to a statistically significant increase of the 12% TCASN. The scores for flavor and texture of the control cheeses were somewhat above those for the experimental cheeses manufactured with starter.

Four strains of lactic acid bacteria, previously isolated from traditional Serra da Estrela cheese and duly identified as Lactobacillus paracasei ssp. paracasei, Leuconostoc mesenteroides ssp. dextranicum, Lactococcus lactis ssp. lactis and Enterococcus faecium, were tested for their aminotransferase, oxidase and dehydrogenase activities towards five amino acids in free form: two branched-chain (leucine and valine), one sulfur-containing (methionine) and two aromatic (phenylalanine and tryptophan) amino acids. For this purpose, both resting cells (RC) and crude cell-free extracts (CFE) were considered; their lyase and demethiolase activities (towards methionine and α-keto-γ-methiolbutyric acid (KMBA), respectively) were also evaluated. Aminotransferase activity (AT) of RC was ca. 10-fold higher towards Met than towards Leu or Val. No AT activity was found in CFE, although these extracts displayed high dehydrogenase activity (DA) towards Phe, especially L. lactis ssp. lactis and E. faecium. In the case of L. paracasei ssp. paracasei and L. mesenteroides ssp. dextranicum, they showed high DA activity, which was ca. 10-fold higher towards Met than towards Leu or Val. E. faecium displayed high oxidase activity towards Met, and produced nine-fold more methanethiol from Met and five-fold more methanethiol from KMBA than the other three isolates under study.