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Production of peptides and free amino acids by two strains of Lactobacillus helveticus in a sterile extract of Reggianito cheese
Simposio; III Simposio Internacional de Bacterias Lácticas; 2009
Institución organizadora:
CERELA (Centro de Referencia de Lactobacilos)
Argentinean hard cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food and can produce a high level of middle-sized and small peptides, as well as free amino acids, during cheese ripening. In this work, we assessed the activity of proteolytic and peptidolytic enzymes of two strains of Lactobacillus helveticus in a sterile substrate prepared with hard cheese. The substrate, a representative model of the aqueous phase of hard-cooked cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90 and 180 days) prepared with the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 mm). Nitrogen content in the extracts was 1.1%; salt concentration and pH were adjusted to 5% and 5.20, respectively. Blanks were prepared by heating aliquots of the extract at 70º C for 10 min in order to inactivate the enzymes. The substrates were incubated at 34ºC during 21 days; samples were taken at 0, 1, 3, 7, 14 and 21 days. Sterility was verified by plating samples on APC agar and incubating at 32 ºC for 48 h. Besides, proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Peptide profiles of blank extracts did not show any changes during incubation, except for a group of hydrophobic peaks. These peaks are characteristic products of the action of the indigenous milk protease, plasmin, and increased slightly during the 21 days of incubation. In the non-heated extracts, great variation in peptide profiles was found as incubation progressed, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the substrate. The substrates prepared with cheeses made with L. helveticus SF138 showed a low production of peptides: the profiles were simpler as incubation progressed and peaks areas diminished. This trend was in agreement with a notable increase in free amino acids content. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, which was evidenced by augmented peaks areas and more complex profiles after longer incubation times. Free amino acids production was lower than in the first case, which suggested that L. helveticus SF209 activity on small peptides was not as intense as that of L. helveticus SF138. This tendency was observed for extracts prepared with cheeses of any age tested. It was concluded that the strains of L. helveticus assayed showed complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. As a consequence, using these strains together in mixed starter cultures would be suitable to increase hard cheese proteolysis and free amino acids production.