A Model to Assess Lactic Acid Bacteria Aminopeptidase Activities in Parmigiano Reggiano Cheese During Ripening

Abstract

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid beta-naphthylamide (betaNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Can the development and autolysis of lactic acid bacteria influence the cheese volatile fraction? The case of Grana Padano

“…The peaks, corresponding to amplicons of specific length on the electropherogram profiles, represent fragments of different sizes, and the areas under the peaks depend on the number of fragments (Suzuki et al, 1998;Lazzi et al, 2004). Each peak, corresponding to amplicons of specific length on the electropherogram profiles, was attributed to bacterial species according to published databases (Lazzi et al, 2004;Gatti et al, 2008aGatti et al, , 2008b, and the areas under the recognized peaks were used in this study to measure the number of the recognized species in the samples. The total area under all of the peaks (sum of attributed and unattributed peaks) of the LH-PCR electropherograms was used in this work to measure the total amount of DNA arising from intact cells.…”

“…Cheeses were sampled from each twin wheel and cut into slices. For each of the samples, one of the four cheeses was analyzed as suggested by Gatti et al (2008aGatti et al ( , 2008b. All of the samples were kept at 4°C after collection and analyzed in a laboratory immediately upon arrival.…”

“…To analyze DNA arising from the whole-cell fraction, samples were prepared as reported by Gatti et al (2008aGatti et al ( , 2008b. Ten grams of grated cheese samples were diluted 1:10 in 20 g/l trisodium citrate (pH 7.5) (Sigma-Aldrich, St. Louis, USA) and homogenized in a blender for 3 min; 1 ml of homogenate was centrifuged and washed using 20 g/l trisodium citrate (pH 7.5).…”

“…Sample preparation for the LH-PCR analysis of the lysed cell fraction was carried out as described by Gatti et al (2008aGatti et al ( , 2008b.…”

Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses

“…A soluble extract that represents the aqueous phase of the food, sterilized by direct UHT method, has been proposed for Cheddar cheese (Crow, Curry, & Hayes, 2001). A Parmigiano Reggiano cheese extract has also been obtained to provide an appropriate culture medium for bacterial species occurring in long ripened cheeses and to assess their aminopeptidase activities (Gatti et al, 2008;Neviani, De Dea Lindner, Bernini, & Gatti, 2009). …”