Effect of the fungal protease EPg222 on the sensory characteristics of dry fermented sausage “salchichón” ripened with commercial starter cultures
Introduction
Proteolysis is one of the most important biochemical changes occurring during the ripening of dry fermented sausages. Protein hydrolysis during ripening yields peptides and free amino acids, which are involved in taste and flavour development (Dı́az, Fernández, Garcı́a de Fernando, de la Hoz, & Ordóñez, 1993; Naes, Holck, Axelsson, Andersen, & Blom, 1995; Ordóñez, Hierro, Bruna, & de la Hoz, 1999). This reaction is catalysed mainly by endogenous enzymes, such as cathepsins and trypsin-like peptidases, but also by proteases produced by micro-organisms involved in the ripening process (Ordóñez et al., 1999; Pezacki & Pezacka, 1986; Selgas, Garcı́a, Garcı́a de Fernando, & Ordóñez, 1993; Toldrá, Rico, & Flores, 1992). Given that endogenous enzymes may be inhibited by salt and curing agents during the ripening process (Rico, Toldrá, & Flores, 1991; Sárraga, Gil, Arnau, Monfort, & Cussó, 1989; Toldrá, Cerveró, Rico, & Part, 1993), several microbial proteases have been assayed to accelerate proteolysis in dry fermented sausages (Dı́az et al., 1993; Dı́az, Fernandez, Garcı́a de Fernando, De la Hoz, & Ordoñez, 1997; Naes et al., 1995; Zapelena, Zalacaı́n, de Peña, Astiasarán, & Bello, 1997; Zapelena, Astiasarán, & Bello, 1999). Although microbial proteases added in the appropriate amount have been reported to be able to increase free amino acids concentration in dry fermented sausages, only a slight increase in the flavour was obtained (Ordóñez et al., 1999). Probably the contribution of micro-organisms added as starter cultures is also necessary to transform free amino acids into volatile compounds.
The role of micro-organisms in the generation of volatile compounds in semi-dry and dry fermented sausages is well documented (Berdagué, Montel, Montel, & Talon, 1993; Montel, Masson, & Talon, 1998; Montel, Reitz, Talon, Berdagué, & Rousset-Akrim, 1996; Stanhke, 1994; Stahnke, 1995). Ethyl esters, methyl aldehydes, methyl ketones, and other volatile compounds in these products have been attributed to lactic acid bacteria and Micrococcaceae (Guo & Chen, 1991; Montel et al., 1996; Stahnke, 1995).
Thus, the effect on flavour development of microbial proteases in dry fermented sausages ripened with starter cultures should be known. The use of proteases obtained from micro-organisms isolated from dry cured meat products could be more appropriate than other ones, since they might be more suited and adapted to function during the ripening process. Protease EPg222 purified from Penicillium chrysogenum Pg222 isolated from dry-cured meat products has a high proteolytic activity against myofibrillar proteins under these conditions of temperature, pH and NaCl concentration in dry-cured meat products (Benito, Rodrı́guez, Núñez, Asensio, Bermúdez, & Córdoba, 2002) and in sterile ripened pork (Benito, Rodrı́guez, Sosa, Martı́n, & Córdoba, 2003).
The aim of this work was to investigate the effect of the protease EPg222 on flavour development in dry fermented sausage “salchichón” when added to commercial starter cultures. In addition, since a negative effect on texture could be expected from the use of a protease, the effect on texture was determined.
Section snippets
Extracellular enzyme
EPg222 is a serine protease obtained from an atoxigenic strain of Penicillium chrysogenum Pg222 isolated from dry-cured ham (Núñez, Rodrı́guez, Bermúdez, Córdoba, & Asensio, 1996). For this assay EPg222 was obtained according with the protocol described by Benito et al., 2002. Purity of the enzyme EPg222 was tested by HPLC and SDS–PAGE (Benito et al., 2002).
Preparation of dry fermented sausages
The mixture for dry fermented sausage “salchichón” was prepared using the following composition: 75% Iberian pork, 25% Iberian pork fat, 1
Results
Microbiological analysis revealed no significant differences between control and enzyme treated samples. Initial counts on MRS and MSA agars were ∼5–6 log CFU/g. Counts in MRS agar reached maximum level above 8 log CFU/g between 3 and 75 days of ripening (Fig. 1). Thereafter they decreased to ∼6 log CFU/g in the sausages by the end of ripening. Counts in MSA agar remained stable at around 5 log CFU/g after 3 days and decreased to ∼4 log CFU/g at 145 days of ripening (Fig. 1). Only yeasts were
Discussion
Addition of protease EPg222 did not affect the evolution of added microbial starter cultures, since no differences in the microbial population were found between control and enzyme added batches.
Use of EPg222 did not either affect the moisture, Aw and pH of dry fermented sausages. In addition, values found in the former parameters are similar to those reported during the ripening process for this kind of product (Garcı́a de Fernando & Fox, 1991; Verplaetse, de Bosschere, & Demeyer, 1989).
The
Acknowledgements
This work was supported by grants from the Spanish Comisión Interministerial de Ciencia y Tecnologı́a (ALI98-0253) and from the Regional Government of the Extremadura (IPR00C042). M. J. Benito was also the recipient of a grant from the Spanish Ministerio de Educación, Cultura y Deporte.
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Present address: Nutrición y Bromatologı́a, Escuela de Ingenierı́as Agrarias, Universidad de Extremadura, Badajoz, Spain.