Abstract
Crustaceans Munida (fam. Galatheideae, ord. Decapodi) were fished in the Southern Adriatic Sea and their proteolytic activities were characterized and tested for potential application in cheese manufacturing. Enzymes extracted from whole crustaceans, mainly serine proteases, showed high caseinolytic and moderate clotting activities. Analysis by 2D zymography of the digestive enzymes extracted from Munida hepatopancreas, showed the presence of several isotrypsin- and isochymotrypsin-like enzymes in the range of 20–34 kDa and 4.1–5.8 pI. Moreover, specific enzymatic assays showed the presence of aminopeptidases and carboxypeptidases A and B. Overall, optimum activity was achieved at pH 7.5 and 40–45 °C. Caseinolytic activity, determined both spectrophotometrically and by SDS gel electrophoresis, indicated higher activity on β-casein than on α-casein. Miniature cheddar-type cheeses and Pecorino-type cheeses were manufactured by adding starter, rennet and Munida extracts to milk. Reverse-phase HPLC and MALDI-ToF mass spectrometry showed a more complex pattern of proteolytic products in cheeses made using Munida instead of chymosin. Munida extracts were found to degrade the chymosin-derived β-casein fragment f193–209, one of the peptides associated with bitterness in cheese. In conclusion, Munida digestive enzymes represent a promising tool for development of new cheese products and shorten cheese ripening when used either alone or in addition to calf rennet.
Highlights
The property of the digestive enzymes from marine organisms to maintain their activity at low temperature might be very useful in food processing in order to avoid bacterial contamination and unwanted chemical reactions [13]
With regard to the application of marine enzyme in the manufacture of cheese, we have introduced the use of the crustaceans Munida
On the basis of preliminary data obtained from the enzyme extracts of whole crustaceans indicating high caseinolytic capability and moderate clotting activity [31], subsequent studies were conducted using the enzymes extracted from the hepatopancreas of the Munida crustaceans, in order to obtain extracts enriched in digestive enzymes only
Summary
Marine enzymes represent a special class of biocatalysts since they are inside organisms living in an environment characterized by high pressure, high salinity, low temperature, little sunlight, i.e., in conditions which are very different from those of the terrestrial environment. The property of the digestive enzymes from marine organisms to maintain their activity at low temperature might be very useful in food processing in order to avoid bacterial contamination and unwanted chemical reactions [13] Serine proteases such as elastase and collagenase are digestive enzymes found in fishes and marine invertebrates such as crab, prawn and lobster [7,10,11]. There is a particular interest in studying proteolytic enzymes from marine species both as possible substitutes for milk-clotting enzymes and for the shortening of cheese ripening times These potential applications are based on the finding that some gastric proteases from marine species may have chymosin-like properties [14,15,16,17,18]. Most interesting in the present context, is the finding that some gastric proteases from marine species have chymosin-like properties [14,15,16,17,18] and may substitute chymosin in milk-clotting activities and shorten cheese ripening times
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