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- Review: Steam distilled spirits from fermented grape pomace Revision: Bebidas destiladas obtenidas de la fermentación del orujo de uvaPublication . Silva, M. L.; Macedo, A. C.; Malcata, F. X.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.
- Microbiological profile in Serra ewes' cheese during ripeningPublication . Macedo, A. C.; Malcata, F. X.; Hogg, T. A.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.
- Peptide hydrolase system of lactic acid bacteria isolated from Serra da Estrela cheesePublication . Macedo, Angela C.; Veira, Marta; Poças, Raquel; Malcata, F. X.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 certi"ed 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.
- Changes in the microflora of Serra cheese: evolution throughout ripening time, lactation period and axial locationPublication . Macedo, Angela C.; Costa, M. Luz; Malcata, F. XavierChanges in the microflora and physicochemical characteristics in Serra da Estrela cheese were examined using a three-way factorial design over a 3.5 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 signtficant 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 statisticalty signrficant variations only throughout the lactation period. Ripening time, lactation period and axial location signtficantly affected the number of yeasts. AN 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 signtficance; 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.
- Assessment of proteolysis and lipolysis in Serra cheese: effects of axial cheese location, ripening time and lactation seasonPublication . Macedo, A. C.; Costa, M. L.; Malcata, F. X.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.
- Purification and characterization of an intracellular aminopeptidase from a wild strain of Lactobacillus plantarum isolated from traditional Serra da Estrela cheesePublication . Macedo, Angela C.; Tavares, Tânia G.; Malcata, F. XavierAn intracellular hydrolase able to cleave Image -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.
- Technological optimization of the manufacture of Serra cheesePublication . Macedo, Angela C.; Malcata, F. XavierIn 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.
- Influence of native lactic acid bacteria on the microbiological, biochemical and sensory profiles of Serra da Estrela cheesePublication . Macedo, Angela C.; Tavares, Tânia G.; Malcata, F. XavierCheesemaking 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.
- Review: Technology, Chemistry and Microbiology of Whey CheesesPublication . Pintado, M. E.; Macedo, A. C.; Malcata, F. X.In whey cheese manufacture, whey, plain or added with milk, is heated by direct fire, bubbling steam or alternatively in jacketed vats. In some cases, salt s or organic acids are previously added. At 80-85 OC, the first particles of curd form; at 85-95 'C, the curd may be cooked for a few minutes to reduce moisture content and/or to obtain the desirable level of browning. After drainage at room temperature during molding for ca. 4 h, whey cheese is stored at ca. 4 'C. The typical mass yield is 6%, but addition of milk, calcium salts and preliminary concentration of protein (by condensation or ultrafiltration techniques) may increase yield considerably. Some types of whey cheeses are supposed to be consumed within a short time upon manufacture (e.g., Ricotta, Requeijdo and Manouri), whereas others bear a longer shelf life (e.g., Gjetost, Mysost and Myzithra). Whey cheeses are significantly different from one another in terms of chemical composition, which is mainly due to variations in the source and type of whey, as well as to the processing practices followed. Moisture content and pH of whey cheeses are usually high and favor microorganism growth (molds, yeasts, lactic acid bacteria and Enterobacteriaceae account for the dominant microflora in these cheeses). Adequate packaging of whey cheeses should be provided, and legislation should be prepared to fix standard characteristics of each type of whey cheese, and hence protect typical products from adulteration and fakes. Marketing efforts should also be aimed at increasing whey cheese consumption, either directly or incorporated in desserts, snack dips and pasta-type dishes.
- Perspectivas científico-tecnológicas na caracterização de melhoramento do Queijo da Serra da EstrelaPublication . Malcata, F. Xavier; Macedo, Ângela C.; Sousa, M. JoséNo intuito de permitir um melhor conhecimento de índole científico-tecnológica sobre o Queijo Serra da Estrela, foi desenvolvido trabalho experimental em duas vertentes principais, a saber caracterização do processo de maturação e melhoramento do processo de fabrico. Tais vertentes abrangem tentativas de descrição de fenómenos de lipólise, proteólise e glicose, bem como tentativas de estabelecimento de relações causa/efeito entre varias tecnologias e as características do produto final, numa perspectiva de racionalização do papel dos diversos microorganismos nativos e do coalho no processo de manufactura do queijo.
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