Angela C. Macedo

Peptide hydrolase system of lactic acid bacteria isolated from Serra da Estrela cheese

Abstract 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.

Influence of native lactic acid bacteria on the microbiological, biochemical and sensory profiles of Serra da Estrela cheese

Cheesemaking from batches of raw ewes 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.

Changes in the microflora of Serra cheese: evolution throughout ripening time, lactation period and axial location

Abstract 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.

Changes in the major free fatty acids in Serra cheese throughout ripening

Abstract Changes in the concentrations of the free fatty acids (FFA) containing an even number of carbon atoms in Serra cheese were monitored using a two-way factorial design over a ripening period of 35 days throughout the cheesemaking season. Both time variables had statistically significant effects at the 5% level on the concentrations of FFA. The concentration of stearic acid increased significantly to 351 mg kg−1 (fat basis) only during the first week of ripening. Concentrations of palmitic and oleic acids increased significantly during all stages of ripening and approximately doubled during the 35-day ripening period to reach levels of 425 and 453 mg kg−1, respectively. Concentrations of butyric, caprylic, lauric and myristic acids were approximately constant during the first week and increased significantly thereafter up to 285, 17, 94 and 59 mg kg−1, respectively, at 35 days. The concentrations of caprylic and myristic acids increased five-fold in 35 days, the concentration of butyric acid increased three-fold, and the concentration of lauric acid almost doubled during the same period. The concentrations of caproic, capric, linoleic and linolenic acids increased significantly to 48, 121, 188 and 126 mg kg−1, respectively, only after 21 days of ripening. The concentration of linoleic acid doubled in that time period, while increases in the concentrations of caproic, capric and linolenic acids were much smaller. The concentrations of butyric, caproic and palmitic acids in cheeses manufactured in February were statistically lower than those in cheeses manufactured in May. The principal FFAs throughout ripening were, according to chain length and degree of saturation, butyric (short-chain), capric (medium-chain), palmitic and stearic acids (saturated long-chain), and oleic (unsaturated long-chain). Saturated and unsaturated long-chain FFA were present at the highest concentration at all stages of ripening. It is concluded that lipolysis in Serra cheese proceeds slowly to total FFA concentration of 2167 mg kg−1 (fat basis) by 35 days of ripening.

Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine sodium caseinates

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.

Esterase activities of intracellular extracts of wild strains of lactic acid bacteria isolated from Serra da Estrela cheese

Abstract 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 of Mineral Concentrations in Serra Cheese during Ripening and Throughout the Cheesemaking Season

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.