Search

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

Bioactive compounds, e.g., protein, polyunsaturated fatty acids, carotenoids, vitamins and minerals, found in commercial form of microalgal biomass (e.g., powder, flour, liquid, oil, tablet, or capsule forms) may play important roles in functional food (e.g., dairy products, desserts, pastas, oil-derivatives, or supplements) or feed (for cattle, poultry, shellfish, and fish) with favorable outcomes upon human health, including antioxidant, anti-inflammatory, antimicrobial, and antiviral effects, as well as prevention of gastric ulcers, constipation, anemia, diabetes, and hypertension. However, scale up remains a major challenge before commercial competitiveness is attained. Notwithstanding the odds, a few companies have already overcome market constraints, and are successfully selling extracts of microalgae as colorant, or supplement for food and feed industries. Strong scientific evidence of probiotic roles of microalgae in humans is still lacking, while scarce studies have concluded on probiotic activity in marine animals upon ingestion. Limitations in culture harvesting and shelf life extension have indeed constrained commercial viability. There are, however, scattered pieces of evidence that microalgae play prebiotic roles, owing to their richness in oligosaccharides-hardly fermented by other members of the intestinal microbiota, or digested throughout the gastrointestinal tract of humans/animals for that matter. However, consistent applications exist only in the dairy industry and aquaculture. Despite the underlying potential in formulation of functional food/feed, extensive research and development efforts are still required before microalgae at large become a commercial reality in food and feed formulation. info:eu-repo/semantics/publishedVersion

The professional work of small animal veterinary staff encompasses a wide diversity of demanding tasks. This has prompted a number of studies covering physical, chemical, biological, ergonomic, or psychological hazards, as well as their health effects upon veterinary workers. However, such results were obtained from self-reported surveys (via paper or online). This study reports the identification of potential hazards and provides a risk assessment of 15 veterinary clinics based on data from walk-through surveys, interviews with workers, and quantification of indoor air quality parameters including concentration of volatile organic compounds (total, isoflurane, and glutaraldehyde). The risk arising from X-ray exposure was unacceptable in seven clinics; X-ray examination should be discontinued in the absence of isolated radiation rooms, poor safety practices, and lack of personal protective equipment. Ergonomic-related hazards and work practices should be revised as soon as possible, considering that improper postures, as well as moving and lifting heavy animals are major causes of musculoskeletal disorders. The risk levels were, in general, small or medium (acceptable) with regard to exposure to physical hazards (such as bites, scratches, cuts, and burns) and biological hazards. It was observed that the indoor air quality parameters including temperature, respirable particulate matter and total volatile organic compounds do not indicate a comfortable workplace environment, requiring clinics' attention to keep the safe environment. The veterinarians and nurses were exposed to isoflurane (above 2 ppm) during surgery if an extractor system for waste gas was used instead of a scavenging system. Finally, veterinary workers did not possess any type of training on occupational safety and health issues, even though they recognized its importance. info:eu-repo/semantics/publishedVersion

Occupational accidents have a major impact upon human integrity, but they also bring about high costs for a country’s social security system. This situation has incidence on the productivity and competitiveness of enterprises, which in Portugal are mostly micro- and small-sized ones. The main goal of this study was to examine the accidents (that were officially recorded) in the decade 1992–2001 in work environments, and working conditions pertaining to the workforce—in attempts to build a picture of the working life situation in that country. Because of incompatibility among data, index numbers by time and by indicator were calculated. The number of occupational accidents decreased from 1992 to 1995, but tended thereafter to stabilize at a still high level; the rate and causes of occupational accidents in Portugal should thus be thoroughly and comprehensively studied, in order to design and implement effective prevention programs. Construction and manufacturing were the most important economic activities in terms of occupational accidents. After 1996, fatal accidents occurred at the highest rates in Lisbon and Tejo Valley’s region, whereas the highest rates of non-fatal ones took place in the north region of Portugal. As expected, the highest numbers of fatal accidents were observed in the 25–44 year group throughout the decade, because this is the age range in which the highest percentage of employment exists. Increasing labor flexibility and overtime working during this decade led to a significant increase of fatal accidents on Saturday and Monday, with a higher incidence in the 12–16 h working schedule. Traumatism with contusion was the major type of injury recorded, and multiple sites was the most frequent description of bodily part injured.

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.

An intracellular hydrolase able to cleave l -lysine-p-nitroanilide was purified from Lactobacillus plantarum strain ESB5004 via two steps of precipitation with ammonium sulfate (at 30 and 50% (w/v)), followed by hydrophobic interaction and ion-exchange chromatographies. The aminopeptidase was purified up to 11-fold, with a final yield of ca. 1%. Its native molecular weight is ca. 70 kDa, and it is apparently composed of two subunits, the molecular weight of which is 34 kDa. The enzyme was assayed using a wide variety of p-nitroanilide (pNA) derivatives as substrates: it hydrolyzed preferentially pNA adducts of hydrophobic and basic amino acid residues; no hydrolysis was in particular observed of Glu-pNA, Gly-pNA or Pro-pNA. The enzyme activity was removed by the metal-chelating agent EDTA, thus suggesting that it is a metallo-enzyme; however, the EDTA-inhibited enzyme was reactivated in the presence of Co2+. Optimal aminopeptidase activity was obtained at 28 °C (pH 7.0) and pH 6.5 (37 °C). The enzyme was inhibited by 10 mM CaCl2 or MgCl2.

The rates and extents of hydrolysis of αS- and β-caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficus-indica, (L.). Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo-first-order enzymatic reaction, in the presence of first-order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet exhibited higher enzymatic efficiency than the fruit extract, irrespective of the source (i.e., bovine, caprine, or ovine) and the type (i.e., αS- or β-casein) of substrate. The enzymatic efficiency (kcat/Km) for αS-casein ranged from 72 to 220 and from 43 to 65 L g-1 h-1, and for β-casein from 242 to 742 and from 55 to 164 L g-1 h-1, for the animal rennet and the enzymatic extract of O. ficus-indica, respectively. Finally, it was observed that β-casein from caprine and ovine caseinates was degraded by O. ficus-indica faster than its αS counterpart, but the reverse was observed for bovine caseinate.

A few European countries have created Appélations d'Origine Protegée (AOP) in order to support legal protection and hence promote enforcement of high-quality standards of selected food products. This paper comprehensively reviews and updates fundamental and applied aspects encompassing tech nological and organoleptic characteristics of AOP traditional cheeses manufactured in the Iberian Peninsula, from ovine and/or caprine milks. Those cheeses from Portugal and Spain can be divided into four distinctive groups, based on milk source and rennet type: ( i) Azeitão, Castelo Branco, Évora, Nisa, Serpa, Serra da Estrela and La Serena cheeses, manufactured with raw ovine milk and coagulated via plant rennet; ( ii) Terrincho, Idiazábal, Manchego, Roncal and Zamorano cheeses, elaborated with raw ovine milk and coagulated via animal rennet; ( iii) Cabra Transmontano and Majorero cheeses, manufac tured with raw caprine milk and coagulated via animal rennet; and ( iv) Amarelo da Beira Baixa, Picante da Beira Baixa and Rabaçal cheeses, manufactured with mixtures of raw ovine and caprine milks and coagulated via animal rennet. Considerable differences between Portuguese and Spanish AOP cheeses exist; the former are, in general, characterized by softer consistencies and similarities to one another in terms of manufacturing protocols (usually encompassing coagulation of plain raw milk followed by slow draining of the curd), whereas the latter are, in general, larger and firmer.

Grape pomace is the solid residue left after juice extraction from grapes, and represents in Mediterra nean countries the most important by-product of the winemaking industry. Steam distillation of fer mented grape pomace will eventually produce a spirit, designated as bagaceira in Portugal, orujo in Spain and grappa in Italy. This paper comprehensively reviews fundamental and applied aspects of the manufacture of these spirits, encompassing their composition as well as metabolic reactions and microbial ecology that determine such composition during fermentation. These spirits adhere to maxi mum levels of methanol (potential toxic compound) and 2-butanol (potential flavor defect) fixed by EC regulations. Available studies pertaining to bagaceira, orujo and grappa have indicated that the final quality of these spirits depends strongly on the quality of the fresh grapes, the storage condi tions, and the distillation equipment and procedure employed.

Lactobacillus paracasei subsp. paracasei ESB 230, Leuconostoc mesenteroides subsp. mesenteroides ESB 136, Lactococcus lactis subsp. lactis ESB 117 and Enterococcus faecium ESB 50, previously isolated from certified Serra da Estrela cheeses, were tested for their aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, dipeptidase and carboxypeptidase activities. The crude cell-free extracts (CFE) of Lb. paracasei ESB 230 exhibited the highest aminopeptidase activity, followed by CFE of Leuc. mesenteroides ESB 136 and, at last, by CFE of L. lactis ESB 117; the aminopeptidase activity in CFE of Ent. faecium was practically non-existent. The four CFE studied also showed appreciable carboxypeptidase activities, although these were lower than their dipeptidase counterparts; in addition, their dipeptidyl aminopeptidase and endopeptidase activities were lower than their aminopeptidase activities. Dipeptides consisting of hydrophobic amino acid residues (i.e. leucine, methionine and phenylalanine) were more rapidly attacked by all CFE than those with hydrophilic amino acid residues. The peptide hydrolase system of CFE of Lb. paracasei ESB 230 was qualitatively quite similar to, but quantitatively more active than that of CFE of Leuc. mesenteroides ESB 136 (except for the endopeptidase); additionally, the CFE of L. lactis ESB 117 and of Ent. faecium ESB 50 were quite distinct from each other, and from the other two CFE tested.

Six different types of bacteria (five strains of lactic acid bacterium and one strain of coliform) isolated from 35-day-old Serra cheese were assayed for proteolytic and lipolytic activities on milk agar and tributyrin agar, respectively. Peptidase and lipase activities were further studied via analytical assaying of free amino acids by spectrophotometry and of free fatty acids by HPLC in in vitro curdled milk (previously prepared from heat-sterilized ovine milk coagulated with thistle flower) subject to ripening for 21 days at 5 °C and 95% relative humidity. Leuconostoc mesenteroides ssp. mesenteroides/dextranicum and Lactococcus lactis ssp. lactis displayed significant proteinase and peptidase activities, where Leuconostoc lactis and Enterococcus faecium only exhibited peptidase activity. Leuc. mesenteroides ssp. mesenteroides/dextranicum, Ent. faecium, and L. lactis ssp. lactis only showed lipase activity on milk fat after a long incubation period. Short- and medium-chain fatty acid residues were released preferentially by microbial lipases, although long-chain fatty acids were also significantly released by lipases from Leuc. mesenteroides ssp. mesenteroides/ dextranicum. Lactobacillus paracasei ssp. paracasei and Hafnia alvei did not display measurable protease, peptidase, or lipase activities. In view of their hydrolase activities, it is suggested that L. lactis ssp. lactis and Leuconostoc spp. (with possible incorporation of Ent. faecium) are a potential mixed-strain starter for Serra cheesemaking.

Seasonal changes of the ash content and mineral concentrations in Serra cheese were studied over a typical 35-day ripening period. Statistically significant differences (at the 5% level) exist between the ash content and the concentrations of Na, K, Ca, P, Mg and Zn in cheeses during ripening. The highest concentrations of Na was obtained in cheese ripened for 7 days, whereas the concentrations of K, Ca, P, Ng and Zn decreased significantly during ripening. For 35-day-old cheeses, concentrations of Na, K and Cu were lowest and concentration of P was highest for cheeses manufactured in May. The concentration of Ca was lowest for cheeses manufactured in February. On average, the most concentrated minerals (in g kg -1 of total solids, TS) in 35-day-old Serra cheese were Na (18.56), Ca (9.70) and P (7.92) and, at a lower level, K (1.70) and Mg (0.96). Only trace levels (in mg kg τs -1 ) of Zn (94.33), Cu (2.26) and Mn (1.25) were detected. A high mineral nutrition quality was thus ascribed to 35-day-old Serra cheese based on the average nutritional densities: 4.8 for Ca, 4.0 for P, 1.1 for Mg, 3.4 for Na, 2.4 for Zn, 0.4 for Cu, 0.2 for Mn and 0.2 for K.

In order to optimize the process of cheesemaking of Serra cheese, the most famous traditional Portuguese cheese manufactured with raw ewes' milk at farm level, several cheeses were produced according to a second-order composite factorial design using temperature of coagulation, amount of plant rennet added and amount of salt added to fresh cheese as the manipulated technological variables. Sensory, microbiological, physicochemical and biochemical analyses were carried out for all experimental cheeses and for two cheeses manufactured by traditional practices. The amount of salt added to fresh cheese affected significantly the sensory, microbiological and biochemical characteristics of 60 day-old Serra cheese, whereas the amount of plant rennet added and the coagulation temperature affected significantly only the sensory and microbiological attributes. None of the three technological parameters at the levels tested played a statistical role at the 5% significance level in the chemical composition of the cheese. It was found that 0.3 g of plant rennet added per litre of milk, a coagulation temperature of 28 °C, and 0.05 g of salt added per cm2 of fresh cheese surface provide the best quality of cheese in terms of highest sensory scores and lowest numbers of viable enterococci and coliforms.

Hydrolysis of the major caseins in Serra cheese manufactured from raw sheep's milk coagulated with a plant rennet (Cynara cardunculus, L.) was monitored by urea-PAGE electrophoresis throughout a 35 day ripening period (with sampling at 0, 7, 21 and 35 days) and throughout the cheesemaking season (with sampling at November, February and May). The αs- and β-caseins were degraded up to 82 and 76%, respectively, by 35 days of ripening. The αs-casein variants (αs2- and αs3-) displayed similar degradation patterns to one another, but different from those of β-casein variants (β1- and β2-). Although the αs-caseins were broken down more slowly than β-caseins at early stages of ripening (97, 95, 80, and 60% of αs2-, αs3-, β1-, and β2-caseins, respectively, were still intact by 7 days), this observation was reversed for later stages of ripening (18, 18, 30, and 20% of αs2-, αs3-, β1− and β2-caseins, respectively, were still intact by 35 days of ripening). The position along the cheese-making season significantly affected the hydrolysis of only the β2- and αs3-caseins. Degradation of αs3-casein was slower in February than in November or May for 21-day old cheeses; cheeses ripened for 7 days or 21 days showed more intact β2-casein when manufactured in May than in November or February. The magnitude of the correlation coefficients pertaining to concentrations of intact αs- and β-caseins indicated that the products of proteolytic breakdown with higher mobility than αs-caseins (tentatively termed α1-I, α2-I, and α3-I) were preferentially correlated with αs-caseins, the products of proteolytic breakdown with mobility between β-caseins and αs-caseins (tentatively termed β1 and β1-I) were preferentially correlated with β1 and β2-caseins rather than with αs-caseins, and the products of proteolytic breakdown with the highest mobility (tentatively termed α β 1 -II and α β 2 -II ) were preferentially correlated with β-caseins.

The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci. Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens, was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.

Scientific evidence associates indoor environment pollutants with respiratory effects (asthma and allergies), and children constitute one of most sensitive groups. Indoor air quality (IAQ) in schools may indeed be a significant health factor for children, with effects on school attendance and performance. Our aim was to characterize IAQ of classrooms in Maia County (north of Portugal) for which there was no information available. The study was conducted in 21 of the 40 primary schools, selected by stratified random sampling. Depending on the dimension, one or two classrooms were tested at each school. Walkthrough surveys of school grounds, buildings, and individual classrooms were done. Continuous measurements were taken of temperature, relative humidity, airborne respirable particles, total volatile organic compounds, and carbon dioxide, whereas bioaerosols were counted on Plate Count Agar during regular school activities. The indoor arithmetic mean for PM10, CO2, TCOV, and bioaerosol concentrations were 0.14 mg/m3, 999 ppm, 0.41 mg/m3, and 4140 UCF/m3, respectively. The values of PM10 and CO2 were close to their acceptable maximum values, but bioaerosols were much higher. This study supports previous studies conducted in school environments and emphasizes the need for proactive indoor air quality audits in school buildings. info:eu-repo/semantics/publishedVersion

Lactococcus lactis subsp. lactis strain ESB110019 and Lactobacillus plantarum strain ESB5004, novel strains that were previously isolated from the wild adventitious microflora of certified Serra da Estrela cheeses, were assayed for esterase activity using, as substrates, ortho - and para -nitrophenyl derivatives of fatty acids. Both strains preferentially hydrolyzed short-chain fatty acids; L. lactis ESB110019 exhibited a stronger esterase activity than Lb. plantarum ESB5004 and cleaved the p -nitrophenyl adducts faster than their o -nitrophenyl counterparts (unlike Lb. plantarum ESB5004).

Changes in the microflora and physicochemical characteristics in Serra da Estrela cheese were examined using a three-way factorial design over a 35 day ripening period, throughout the lactation period, and for several slices taken normal to the cheese axis. Lactic acid bacteria were the major components of the microflora during ripening throughout the lactation period, but no statistically significant differences were detected between the surface and the interior of the cheese. Both enterobacteriaceae and staphylococci in the curd were at maximum numbers during the first week and were still at significant numbers towards the end of the maturation period. The numbers of staphylococci were statistically different between slices and at different times within the lactation period, whereas the numbers of enterobacteriaceae showed statistically significant variations only throughout the lactation period. Ripening time, lactation period and axial location significantly affected the number of yeasts. All factors studied had statistically significant effects on pH, salt and moisture content; the hypotheses that ripening time, lactation period, and axial location had effects on salt and moisture contents were accepted, in this order, at increasingly lower levels of significance; the hypotheses that ripening time, axial location, and lactation period had effects on pH were accepted, in this order, at increasingly lower levels of significance.

Summary - The degrees of proteolysis and Iipolysis in Serra cheese were measured in cheese samples obtained from experiments laid out as a three-way facto rial design replicated twice. The independent variables studied were the location within the cheese (from the center to the surface), the position in the lactation season (from October to June) and the ripening time (from 0 to 35 days). Ali three variables had statistically significant effects on the concentration of water soluble peptides (WSP), but only the ripening time and the period within the lactation season affected the fat acidity (FA). The extents (alter the given ripening period) and the rates (averaged over the ripening period) of generation of WSP and FA were highest for spring and lowest for autumn. The lowest values for both the proteolysis extent and the proteolysis rate were obtained for the rind. The rates of proteolysis and lipolysis tended to decrease with ripening time; most of the lipolysis occurred during the first week, but proteolysis was still in progress by 35 days of ripening.