ORIGINAL PAPER Mo Ânica Flores ? M. Concepcio Ân Aristoy ? Fidel Toldra  Curing agents affect aminopeptidase activity from porcine skeletal muscle Received: 11 February 1997 / Revised version: 25 April 1997 AbstractmThe effect of curing agents (salt, nitrite, nitrate, ascorbic acid, glucose and polyphosphates) on the main pure aminopeptidases involved in the release of amino acids during the processing of cured meat products has been studied. Salt strongly inhibited alanyl aminopeptidase and pyroglutamyl aminopeptidase: about 40±50% of their original activities were recovered in the presence of 4±5% salt, the typical concentration in dry-cured meat products, while leucyl aminopeptidase was not affected. However, arginyl aminopeptidase was activated to 2±3 times its control level. Nitrate and nitrite did not affect aminopepti- dase activity. Ascorbic acid (500 mg/l) slightly inhibited arginyl, alanyl and pyroglutamyl aminopeptidases whereas glucose (20 g/l) activated (to almost 5 times its control level) leucyl aminopeptidase activity. Polyphosphates in- hibited (25%) alanyl and arginyl aminopeptidases. Key wordsmAminopeptidase ? Muscle exopeptidases ? Curing agents Introduction Aminopeptidases are important enzymes in the develop- ment of the characteristic flavour of meat products, due to large generation of free amino acids during their processing [1, 2]. This increment is produced by the action of proteo- lytic enzymes such as aminopeptidases [3, 4] which are capable of hydrolysing amino acids from the N-terminus of peptides and proteins [5]. Aminopeptidases have been found in the cytosolic fraction of skeletal muscle [6±8] and in other tissues [9±12]. Four aminopeptidases have been distinguished in the cytosolic fraction of skeletal muscle. First, alanyl amino- peptidase (AAP), which accounts for as much as 83% of the total aminopeptidase activity and has a broad substrate specificity towards aromatic, aliphatic and basic amino- acyl-bonds [4, 13, 14]. Second, arginyl aminopeptidase (RAP) or aminopeptidase B [15] represents 11% of the total aminopeptidase activity [16, 17] and has been char- acterized as a chloride-activated enzyme hydrolysing basic termini [4, 18]. Third, leucyl aminopeptidase (LAP) is a zinc metalloenzyme with an alkaline optimal pH that hydrolyses leucine and other hydrophobic amino acids [19] and represents 3% of the total aminopeptidase activity [20]. Fourth, pyroglutamil aminopeptidase (PGAP) also accounts for 3% of the total aminopeptidase activity and catalyses the release of the N-terminal pyroglutamil group at basic pH [17, 20]. Aminopeptidase activities have been reported as being present in muscle and adipose tissue from both raw and dry- cured ham and have shown good stability even after 8 months of curing [21]. However, their role during the processing procedure has not been elucidated because the measurement of the aminopeptidase activities in muscle extracts suffers from interference because of the absence of specific substrates for each aminopeptidase. In fact, this was shown to be the case in a previous work [22] which studied the effect of curing agents on the hydrolysis of amino acids. In this paper the effect of curing agents on the main purified muscle aminopeptidases is shown in order to gain a better understanding of the contribution made by each particular aminopeptidase to the total proteolytic action. Materials and methods Materials. Aminoacyl-7-amido-methyl coumarin substrates (aac- AMC) were obtained from Sigma (St. Louis, Mo., USA). HPLC column PL-1000 SAX (50 ´ 5 mm, 8 mm particle size) was purchased from Hewlett-Packard (Palo Alto, Calif., USA). Enzyme purification was done using a biocompatible (titanium) 1050 Hewlett-Packard liquid chromatograph equipped with a variable wavelength UV detec- tor (280 nm). Muscles (biceps femoris) from 6-month-old pigs were taken just within 1 h of death and immediately cooled to below 4 °C. M. Flores ? M. C. Aristoy ? F. Toldra Â( ) Instituto de Agroquõ Âmica y Tecnologõ Âa de Alimentos (CSIC), Apt. 73, E-46100 Burjassot, Valencia, Spain Z Lebensm Unters Forsch A (1997) 205: 343 ± 346 Ó Springer-Verlag 1997