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Annals of Microbiology, 54 (3), 269-281 (2004)

Evaluation of proteolytic activity of Staphylococcus xylosus strains in Soppressata Molisana, a typical Southern Italy fermented sausage

F. NAZZARO1, A. DI LUCCIA1,2, P. TREMONTE3, L. GRAZIA4, E. SORRENTINO3, L. MAURELLI1, R. COPPOLA3*

1Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche, Via Roma 52, 83100 Avellino; 2Dipartimento di Produzione Animale, Università degli Studi di Bari, Via G. Amendola, 70126 Bari; 3Dipartimento di Scienze e Tecnologie Agro-Alimentari, Ambientali e Microbiologiche, Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso; 4Dipartimento di Protezione e Valorizzazione Agroalimentare, Via F.lli Rosselli 107, 42100 Reggio Emilia, Italy

Abstract - Two Staphylococcus xylosus strains were separately used as starter cultures in Soppressata Molisana, a typical Southern Italy fermented sausage, with the aim to obtain an overview of the role of microbial proteolysis. Microbial counts, pH trends and soluble N (NPN, peptide and total amino acid content) were monitored throughout the ripening (26 days) and at the end of storage (94 days) in pork fat or under vacuum packaging. A lower production of soluble N was observed in the traditional batch and in products inoculated with non-proteolytic Staphylococcus xylosus strain. Moreover, staphylococci growth was less vigorous than that observed in the sample inoculated with the proteolytic strain. The results show that the proteolytic phenomena, ascribable to bacterial activity, can take on a meaningful role when the food matrix has been colonised by strains having powerful proteolytic capacities. In fact, the increasing trend in NPN/NT, small peptides and total free amino acids, shown in batch inoculated with proteolytic Staphylococcus xylosus can be justified by a considerable proteolytic and peptidasic capacity exhibited by the strain added as starter. This work could contribute to define the microbiological role of proteolysis occurring in fermented sausage. Key word: proteolysis, Staphylococcus xylosus, fermented sausage, microbial starter.

INTRODUCTION The need to store meat for long periods induced the generation of many types of products, which diversified in different countries and geographical areas according to usage and customs. Among these products, fermented sausages are widespread. Italy well reflects this pattern, since many different types of dry sausages are produced throughout the various regions of the country. Sop* Corresponding Author. Phone: +39-0874404870; Fax: +390874404652; E-mail: [email protected]

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pressata Molisana stands as a fine example of these dry fermented sausages. It is a typical Southern Italy product particularly appreciated for its sensorial characteristics, derivable by a brief but intense ripening period in which microbial flora plays an important role. In the initial phase of artisanal sausage manufacture, pork meat and fat are minced and the handling of the fresh product enriches it of natural microflora. Afterwards, during ripening and ageing, the most important biochemical event is represented by enzymatic protein hydrolysis (Artiasaran et al., 1990; Roca and Ince, 1990; Garcia de Fernando and Fox, 1991; Sarra et al., 2004), an event that is mostly responsible for the formation of small peptides and free amino acids. Free amino acids are the main precursors of volatile compounds and concur to the taste of the final product (Aristoy and Toldrà, 1995; Toldrà and Verplaetse, 1995). Some of these molecules (2 and 3-methylbuthanal, 2 and 3methylbuthanol, and 2-methylpropanal) are, in fact, considered as fundamental and peculiar flavour components in fermented sausages (Berdaguè et al., 1993; Stahnke, 1995a, 1995b; Montel et al., 1996; Moller et al., 1998). Nowadays, starter cultures or their proteolytic enzymes are mainly used to shorten ripening (Blom et al., 1996; Hagen et al., 1996; Diaz et al., 1997), to enhance the flavour (Talon et al., 1998; Stahnke, 1999; Luongo et al., 2001), and to improve the safety of the product (Hernandez et al., 1993; Lucke, 2000). The species used as starter cultures belong mainly to the genera Lactobacillus, Micrococcus, Kocuria, Staphylococcus and Pediococcus. The role of microbial proteases and peptidases during the ripening of dried sausages is still being debated: Verplaetse et al. (1992) attributed an important role to the microbial enzymatic activities during proteolysis, Hughes et al. (2002) supported that the bacterial peptidases contribute significantly to the release of free amino acids. However, other authors (Hierro et al., 1997; Molly et al., 1997; Kenneally et al., 1999) stated that proteolysis is due to endogenous enzymes and that microbial flora plays a minor role, if any, in this phenomenon. This work could contribute to define the microbiological role of proteolysis occurring in fermented sausage by comparing and evaluating the changes in proteolysis products in Soppressata Molisana samples produced with different Staphylococcus xylosus strains.

MATERIALS AND METHODS Sausage processing. Soppressata Molisana was produced in a small plant using traditional technologies (Coppola et al., 1997, 1998). The mix was prepared with minced lean pork meat (longissimus dorsi and psoas), fat (2%), glucose (0,3%), and KNO3 (0,02%). It was then divided into three batches: the first (batch C) was not inoculated and was utilised as control, while the remaining batches (batches P+ and P­) were inoculated with two different starter cultures, each belonging to Staphylococcus xylosus species and previously isolated from Soppressata Molisana manufactured without the addition of starter cultures (Coppola et al., 1997). Batch P+ was inoculated with Staphylococcus xylosus strain XC1, characterised by a well-known proteolytic activity (previously tested in milk) (Coppola et al. 1997). Batch P­ was prepared with a non-proteolytic S. xylosus strain (XO3).

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The strains were grown in MS broth (Oxoid) for 48 h, harvested by centrifugation, washed in sterile quarter-strength Ringer's solution and used at a final concentration of 106 UFC/g. After filling into artificial casings, the products were kept at 2 °C for 12 h, pressed for 24 h at room temperature and dried for 7 days in store rooms (with relative humidity increasing from 60 to 80% and temperature decreasing from 18 to 11 °C). Subsequent ripening phase was carried out for 26 days at 80% relative humidity and 11 °C. At the end of the ripening, batches were store at about 11 °C under vacuum packaging or in pork fat. Three different samples from each batch were analysed immediately after filling, at 3, 10, 19 and 26 days of ripening and after three months of storage. Bacterial count and pH measurement. Ten g from each sample were aseptically withdrawn and homogenised with sterile quarter-strength Ringer's solution in a Lab-blender Stomacher (Seward Medical, London, UK). Micrococcaceae were counted on Mannitol Salt Agar (Oxoid) after incubation at 28 °C for 36 h. Lactic Acid Bacteria were determined on MRS agar (Oxoid) after anaerobic incubation (Gas Pack Anaerobic System, BBL) at 28 °C for 72 h. The potentiometric measurement of pH was performed by inserting the pin electrode of a pH-meter (Radiometer, Copenhagen, Denmark) into each sample. Peptide analyses. Peptide profiles of protein extracts were analysed by Reversed Phase High Performance Liquid Chromatography (RP-HPLC) by using a Gold System 126 Apparatus (Beckman, CA, USA). Samples were treated according to Fadda et al. (1999a) modified as follows: each protein extract was deproteinised with acetonitrile (1:2.5 v/v), placed at 4 °C for 1 h and centrifuged at 13,000 rpm for 15 min. The supernatant, containing peptide fraction, was concentrated by evaporation to dryness in a CHRIST ALPHA RVC (B-Braun Biotech International, Melsungen, Germany) and dissolved in 20 µL of 0.01% v/v trifluoroacetic acid in Milli Q water (solvent A). Twenty µL of deproteinised samples diluted 1:5 in solvent A were applied onto a Khromasyl C18 column (Akzo Nobel, Sweden). The elution system consisted of solvent A and solvent B (acetonitrile-water 60:40 with 0.01% v/v trifluoroacetic acid). The elution was performed at 0.7 mL/min flow rate isocractically in 5% solvent B for 5 min, with a linear gradient from 5 to 60% of solvent B for 35 min, then finally to 100% solvent B for 10 min. Peptides were detected at 214 nm by an UV detector 166 (Beckman, CA, USA). The absolute area of peaks was integrated by using software Gold System 126. Amino acid analyses. 1,5 mL from each deproteinised sample obtained as previous described were lyophilised and dissolved in 500 µL of deionised water. Fifty µL of 1M borate buffer pH 6.2 and 250 µL of 15 mM fluorenylmethyl chloroformate (FMOC) were added to 200 µL of sample. After 40 s, the mixture was extracted with 1 mL pentane (Einarsson et al., 1983). The extraction was performed in duplicate. Acetic buffer was prepared by adding 3 mL glacial acetic acid and 1 mL triethylamine to 1 L of distilled water and adjusting the pH value to 4.2 with NaOH. Amino acid profiles of deproteinised extracts were analysed by HPLC with a Gold System 126 and an Analog Interface Module 406 (BeckAnn. Microbiol., 54 (3), 269-281 (2004) 271

man, CA, USA). The fluorescence detector consisted of a Shimadzu RF-55 ( excitation 260 nm; emission 313 nm). Samples were diluted 1:20 with solvent A (acetonitrile: methanol: acetic buffer 10:40:50) and applied onto an Spherisorb S3 ODS2 4.6x150 mm (Waters). The elution varied from solvent A to solvent B (acetonitrile: acetic buffer 50:50). The gradient was started after 3 min from the injection. NPN. To determine the nitrogen fraction, 30 g of each sample were homogenised with distilled water to a final volume of 350 mL, incubated at 4 °C for 1 h and then centrifuged (6,500 rpm for 6 min) by a Beckman Avanti TM J-25. The pellet was re-extracted with 100 mL of distilled water and re-centrifuged as described by Diaz et al. (1993). The two supernatants represented the water-soluble nitrogen fraction (WSN). An aliquot of this fraction was mixed (1:1 v/v) with 25% trichloracetic acid (Carlo Erba, Milano, Italy), incubated at 4 °C for 30 min and then filtered using Whatman n. 1 filters. Total nitrogen and non-protein nitrogen were determined by the Kjeldahl method (BUCHI, Switzerland).

RESULTS Microbial flora The trend of Micrococcaceae during the ripening and the storage is reported in Fig. 1. After 10 days of ripening an appreciable increase of this microbial group was observed in all batches, but the addition of the starter ensured an important growth from the first days of ripening, with a maximum level after 10 days, wherease the uninoculated batch reached the highest level only after 19 days. Decrease characterised the ripening of all products until day 26. During the storage, a substantial stability was found in counts from both batches C and P+, whereas in batch P­ a novel increase was noted. Probably are to the ability to grow at the storage temperature of the strain XO3. Trend of Lactic Acid Bacteria (LAB) during the ripening and the storage is reported in Fig. 2. A fair increase of LAB was observed during the initial step of the ripening in all batches, with particular reference to batch P+. Growth was observed until the 19th day of ripening, while a decrease characterised all batches from the 19th to the 26th day of ripening. During storage, batches P­ and C were characterised by a further decrease, while counts from batch P+ were almost stable relatively to the samples stored in pork fat. pH In all batches a sensible decrease in pH was observed during the first days of ripening (Fig. 3), with the lowest values after the 10th day. The decrease in batch P+ was more evident (0.67 units) than that observed in batches C e P(0.11 and 0.23 units respectively). From the 19th day of ripening, all batches were characterised by an increase in pH values. Non-protein nitrogen (NPN) Figure 4 reports the NPN percentage on the total nitrogen for the three batches investigated during the periods of ripening and storage. Batches C e P­

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FIG. 1 ­ Changes in counts of Micrococcaceae during the ripening and the storage of soppressata molisana.

FIG. 2 ­ Changes in counts of Lactic Acid Bacteria during the ripening and the storage of soppressata molisana.

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( ) control, batch without starter inoculation and stored under pork fat; ( ) control, batch without starter inoculation and stored under vacuum; ( ) batch inoculated with proteolytic S. xylosus XC1 and stored under pork fat; () batch inoculated with proteolytic S. xylosus XC1 and stored under vacuum; ( ) batch inoculated with non proteolytic S. xylosus XO3 and stored under pork fat; () batch inoculated with proteolytic S. xylosus XO3 and stored under vacuum.

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FIG. 3 ­ Trend of pH during the ripening and the storage of soppressata molisana.

FIG. 4 ­ Changes in NPN/NT ratio during the ripening and the storage of soppressata molisana.

( ) control, batch without starter inoculation and stored under pork fat; ( ) control, batch without starter inoculation and stored under vacuum; ( ) batch inoculated with proteolytic S. xylosus XC1 and stored under pork fat; () batch inoculated with proteolytic S. xylosus XC1 and stored under vacuum; ( ) batch inoculated with non proteolytic S. xylosus XO3 and stored under pork fat; () batch inoculated with proteolytic S. xylosus XO3 and stored under vacuum.

showed a slight but constant increase in NPN/NT ratio through the 26 days of ripening, summing up to a 15% increase in comparison to the initial value. During storage, these values remained substantially stable. The picture for batch P+ was different: there was a 19% increase in NPN/NT ratio, compared to the starting values, already at the 10th day of ripening. These values decreased at the 19th day and then increased again at the 26th day, averaging out to a 30% increase in comparison to the starting value. For this batch, a further increase was observed during storage, reaching a total of about 36% increase at the end of the period compared to the starting value. Peptide formation The most significant peak variations in peptide chromatographic profiles are reported in Fig. 5. They are expressed in terms of absolute area. To describe proteolytic phenomena, we took into account the peaks showing substantial differences among the batches, in particular peaks eluted within the 2nd and the 3rd minute (arbitrarily labelled as peak 1), the 5th and 7th minute (labelled as peak 2) and finally the peak eluted at the 15th minute (peak 3). The first two peaks

Absolute area (mm2)

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Absolute area (mm2)

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FIG. 5 ­ Variation in peptide chromatographic profiles during ripening and storage of soppressata molisana; peak 1: peptide eluted within the 2nd and the 3rd minute; peak 2: peptide eluted within the 5th and the 7th minute.

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contained the most hydrophilic components, among which were low-weight amino acids and peptides. The third peak, eluted at a later time, was likely composed of greater and more hydrophobic peptides, or of aromatic amino acids, as reported by Bottiglieri et al. (2000). For batches C and P­ the area values of peak 1 increased steadily until the 26th day of ripening, and subsequently showed a constant trend during storage. The whole area of peak 1, in fact, averaged 590 mm2, reaching about 1000-1100 mm2 at the end of ripening (26 days) and maintaining these values also at the end of storage. Batch P+, instead, showed, in corrispondence to peak 1, an increasing trend until the 10th day of ripening, out of which the average increase in area was about 700-1300 mm2. From the 10th to the 19th day, we recorded a decrease in absolute area of peak 1 down to 1100 mm2. In the time between the 19th and the 26th day in the same batch, the trend began to increase again, ultimately reaching about 1450 mm2. Batch P+ showed a further increase in peak 1 values during storage, averaging about 1650 mm2 at the end of this phase. Peak 2 showed an increase for all batches in the first 10 days of ripening. At the 10th day the highest area values were reached for batches P+ and P­ (420 and 248 mm2, respectively), while for batch C the maximum value (90 mm2), was reached only at the 19th day. At the end of the ripening, peak area values decreased in all batches. During storage, peak 2 increased for batch P-, while it was substantially constant in control (C) and in P+ batches. The third peak had a different trend for the three batches. It was present in batch C during all the ripening, while, for the inoculated batches, it was no longer present already by the 3rd day (P­) and by the 10th day (P+) (data not shown). The three peaks showed similar variations in both storage systems. Amino acids The content in total amino acids (Fig. 6) showed a similar trend throughout ripening for all batches. In the first three days of ripening, there was a steady increase, especially for batch P+, which showed a sensitive increase in the concentration of amino acids, from 1270 µg/mL to 6000 µg/mL in comparison to batches P­ and C, which reached a 4200 µg/mL concentration level of amino acids at the 3rd day while starting off from the same values. However, batch P+, from the 3rd day, gave constantly higher values in comparison to those from batches P­ and C. From the 3rd to the 10th day, a sensitive decrease was observed in the concentration of amino acids, while, at the 19th day it slightly increased, and finally it markedly increased at the end of ripening. Batch P+ revealed a constant increase in amino acids, with a final value of 9000 µg/mL recorded at the end of ripening (26 days); the increase recorded for batches C and P­ was less substantial, reaching amino acid concentration levels of 47004900 µg/mL. During storage, whether in pork fat or vacuum-packed, it was possible to note a further increase in free amino acid levels in batch P+, whereas batches C and P­ were characterised by a rather constant trend.

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13000 12000 11000 10000 Amido acid (µL/mL) 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 10 20 30 40 50 60 70 80 90 100 110 120 130

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FIG. 6 ­ Trend of free amino acid concentration during ripening and storage of soppressata molisana: ( ) control, batch without starter inoculation and stored under pork fat; ( ) control, batch without starter inoculation and stored under vacuum ; ( ) batch inoculated with proteolytic S. xylosus XC1 and stored under pork fat; () batch inoculated with proteolytic S. xylosus XC1 and stored under vacuum; ( ) batch inoculated with non proteolytic S. xylosus XO3 and stored under pork fat; () batch inoculated with proteolytic S. xylosus XO3 and stored under vacuum.

DISCUSSION The role played by Micrococcaceae in the proteolytic and peptidasic phenomena in meat products has yet to be fully understood. Several authors (Chen and Guo, 1992; Bacus, 1996) reported that species traced to this family have a fundamental role in the mentioned activities and are responsible for a steady increase in free amino acids. Selgas et al. (1986) found in their in vitro studies that micrococci strains isolated from dry fermented Spanish sausages are capable of intra- and extracellular proteolytic activities. The inability for proteolytic action and a low aminopeptidasic activity was found by Molina (1992) and Montel et al. (1996) in studies on species from the genus Staphylococcus. Recent studies carried out by Bover-Cid et al. (2001) evidenced that the use of proteolytic staphylococci as starter determines a considerably strong increase in non­protein nitrogen and total free amino acids. We evaluated and compared the proteolytic activity of two different strains of S. xylosus during ripening and storage of Soppressata Molisana. The correlation between microbiological and biochemical results allows to

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better understand the probable role held by proteases and peptidases both endogenous and of bacterial origin. Peptide production, which characterises the process of ripening, is particularly tied to bacterial growth and to the NPN/NT ratio. In particular, the trend observed in peak 1 area shows a strict correlation (r=0,9) with the variation of non-protein nitrogen content (NPN). Furthermore, it is worth noting the good correlation (r=0,89) between the variations in Micrococcaceae counts during ripening and the trend in area of peak 2. It is therefore possible to consider peak 1 and peak 2 as reliable markers for trends in non-protein nitrogen and for the variation in Micrococcaceae counts, respectively. Combining the values related to NPN/NT ratio with the variation of total free amino acids, chromatographic analyses and Micrococcaceae counts, it is possible to build an intricate network which can describe the proteolytic pathways that take place during the ripening and the storage of Soppressata Molisana. The life of the foodstuff can be divided into four phases, each affected by different parameters considered herein. During the first 10 days of ripening, constituting the first phase of the product's life and including the exponential growth phase of Micrococcaceae, proteolytic phenomena are practically controlled by endogenous proteases, as widely reported in literature (Toldrà et al., 1993; Verplaetse 1994; Molly et al 1997; Hierro et al., 1997, 1999; Lucke, 2000). Nevertheless, throughout of this first phase, the action of proteolytic strain S. xylosus is already noteworthy. For all batches, and particularly for batch P+, in this period a constant increase in NPN/NT values can be observed, an event, which perfectly correlates itself to the increase in the concentration of small peptides. The trend in the concentration of total free amino acids is in agreement with the microbiological and biochemical events, which characterise this phase. The increase of total free amino acids, occurring in the first three days of ripening, is essentially traceable to endogenous peptidasic activity and to an initial concurrent proteolytic activity from the strain used as starter in batch P+; its decrease from the 3rd to the 10th day is attributable to their assimilation by microflora, as described in literature (Fadda et al., 1999b). This supports our thesis, which states that the increase in the concentration of small peptides, to be traced to meat peptidasic activities, strongly influences the increase in non-protein nitrogen during the first days of the ripening. Nevertheless, the picture can change and the proteolytic phenomena, ascribable to bacterial activity, can take on a meaningful role when the food matrix has been colonised by strains having powerful proteolytic capacities. Only the presence of proteolytic activity in S. xylosus XC1 can justify the more intense increase in NPN/NT, detected in batch P+ from the 10th day of ripening. Moreover, the high value of NPN/NT could justify the growth of LAB revealed in batch P+ and probably due to the greater availability of easily assimilable nitrogen derived from the peptidasic activities of S. xylosus strain. In fact, the growth of LAB was significantly lower in the batches C and P­. Between days 10 and 19, the batches showed variable behaviour of different parameters. Batch P+ showed a decrease in NPN/NT values that seems to be correlated with the trend of peak 1. This decrease is, in part, owed to the decline in the growth of Micrococcaceae and partially to the assimilation of watersoluble nitrogen by LAB, which showed in this period the highest value. In the

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same batch, we observed an increase in the levels of total free amino acids, not observed in the batches C or P­. This phenomenon is likely attributable to the presence of a microbial peptidasic activity of Lactobacillus sakei (Montel et al., 1995), a dominating species among the LAB present in this type of product (Coppola et al., 1998). Batches C and P­ behaved similarly during ripening and showed a slight increase in NPN/NT values, still again correlated to peak 1 and to the level of free amino acids. Both increases could be ascribable to a prevailing activity of endogenous enzymes. In the third phase (from the 19th to the 26th day of ripening), the role of Staphylococci in proteolytic pathways becomes more and more important. By a careful consideration of the results obtained regarding NPN/NT, P+ values were recorded constantly higher than those of batches P­ and C. This increase, which was especially evident in batch P+, is probably due to the bacterial peptidases released by cell lysis and can be also deduced from the increasing trend of the total free amino acids, suggesting a predominant bacterial peptidases activity together with the endogenous one. In the fourth phase, corresponding to the storage period, batches C and P­ were characterised by a substantially constant trend of both NPN/NT and of the level of free amino acids. The different increasing trend shown in batch P+, can be justified by a considerable proteolytic and peptidasic capacity exhibited by the strain added as starter, which surprisingly showed new growth in this last phase. Batches C and P­, though having a intense growth of Micrococcaceae trend did not show a correlate increase in either NPN/NT values and free amino acid levels during the three phases of the ripening nor in the fourth phase of storage. These evidences add more significance to the results obtained in the present work highlighting the activities expressed by the bacteria naturally involved in the production and in the ripening of this kind of product as well as those expressed by single strains, added as starter, and able to significantly influencing the quality of products.

REFERENCES Aristoy M., Toldrà F. (1995). Isolation of flavours peptide from raw pork meat and drycyred ham. In: Charalambous G., Ed., Food Flavours: Generation Analysis and process Influence. Elsevier Science, Amsterdam, pp. 1323-1344. Artiasaran I., Villanueva R., Bello J. (1990). Analysis of proteolysis and protein insolubility during the manufacture of some varieties of dry sausage. Meat Sci., 28: 111117. Bacus, J. N. (1986). Fermented meat and poultry products. In: Pearson A.M., Dutson T.R., Eds, Advances Meat and Poultry Microbiology. Macmillan, Loudres, pp. 123164. Berdagué J.L., Monteil P., Montel M.C., Talon R. (1993). Effects of starter cultures on the formation of flavour compounds in dry sausage. Meat Sci., 35: 275-287. Blom H., Hagen B.F., Pedersen B.O., Holck A.L., Axelsson L., Naes, H. (1996). Accelerated production of dry fermented. Meat Sci., 43: 229-242. Bottiglieri C., Scaloni, A. Fedele, E. Romano, R. Bergamo, P., Di Luccia, A. (2000). Caratterizzazione della frazione idrosolubile inferiore a 300 Dalton isolata da prosciutti crudi e cotti di carne suini. In: Porretta, S., Ed., Ricerche e Innovazioni dell'Industria Alimentare. Chiriotti, Pinerolo, Italia, pp. 583-591.

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Bover-Cid S., Izquierdo-Pulido M. and Vidal-Carou M.C. (2001). Effect of the interaction between a low tyramine-producing Lactobacillus and proteolytic staphylococci on biogenic amine production during ripening and storage of dry sausages. Int. J. Food Microbiol., 65: 113-123. Chen, M.T., Guo, S.L. (1992). Studies on the microbial flora of Chinese-style sausage. II. Action of selected organism isolated from Chinese-style sausage on porcine muscle proteins. Fleischwirtsch, 72: 1126-1132. Coppola R., Iorizzo M., Saotta R., Sorrentino E., Grazia L. (1997). Characterization of Micrococci and Staphylococci isolated from Soppressata Molisana, a Southern Italy fermented sausage. Food Microbiol., 14: 47-53. Coppola R., Giagnacovo B., Iorizzo M., Grazia, L. (1998). Characterization of lactobacilli involved in the ripening of Soppressata Molisana, a tipical Southern Italy fermented sausage. Food Microbiol., 15: 347-353. Diaz O., Fernandez M., de Fernando, G.D.G., de la Hoz L., Ordonez, J.A. (1993). Effect of the addition of pronase E on the proteolysis in dry fermented sausages. Meat Sci., 34: 205-216. Diaz O., Fernandez M., de Fernando G.D.G., de la Hoz, L., Ordonez, J.A. (1997). Proteolysis in dry fermented sausages - The effect of selected exogenous proteases. Meat Sci., 46: 115-128. Einarsson A., Josefsson B., Lagerkvist, S. (1983). Determination of amino acids with 9fluorenylmethylchloroformate and Reversed-Phase High-Performance Liquid Chromatography. J. Chromatog., 282: 609-618. Fadda, S. Sanz, Y. Vignolo, G. Aristoy, M. Oliver, G., Toldra, F. (1999a). Characterization of muscle sarcoplasmic and myofibrillar protein hydrolysis caused by Lactobacillus plantarum. Appl. Environ. Microbiol., 65: 3540-3546. Fadda S., Sanz Y., Vignolo G., Aristoy M., Oliver G., Toldra, F. (1999b). Hydrolysis of pork muscle sarcoplasmic proteins by Lactobacillus curvatus and Lactobacillus sake. Appl. Environ. Microbiol., 65: 3578-3584. Garcia de Fernando G.D., Fox P.F. (1991). Study of proteolysis during the processing of a dry fermented pork sausage. Meat Sci., 30: 367-383. Hagen B.F., Berdauge J., Holck A., Naes H., Blom H. (1996). Bacterial proteinase reduces maturation time of dry fermented sausages. J. Food Sci., 61:1024-1029. Hernandez P.E., Rodriguez J.M., Cintas L.M., Moreira W.L., Sobrino O.J., Fernandez M.F., Sanz B. (1993). Utilization of lactic bacteria in the control of pathogenic microorganisms in food. Microb. Spanish Review, 9: 37-48. Hierro E., de La Hoz L., Ordonez J.A. (1997). Contribution of microbial and meat endogenous enzymes to the lipolysis of dry fermented sausages. J. Agr. Food Chem., 45: 2889-2995. Hierro E., de La Hoz L., Ordonez J.A. (1999). Contribution of the microbial and meat endogenous enzymes to the free amino acid and amine contents of dry fermented sausages. J. Agr. Food Chem., 47: 1156-1161. Hughes M.C., Kerry J.P., Arendt E.K., Kenneally P.M., McSweeney P.L.H., O'Neill E.E. (2002). Characterization of proteolysis during the ripening of semi-dry fermented sausages. Meat Sci., 62 (2): 205-216. Kenneally P.M., Fransen N.G., Grau H., O'Neill E.E., Arendt E.K. (1999). Effects of environmental conditions on microbial proteolysis in a pork myofibril model system. J. Appl. Microb., 87: 794-803. Lucke F. K. (2000). Utilization of microbes to process and preserve meat. Meat Sci., 56: 105-115. Luongo D., Giagnacovo B., Fiume I., Iorizzo M., Coppola R. (2001). Volatile compounds in "soppressata molisana" style salami fermented by Lactobacillus sakei. Ital. J. Food Sci., 13: 19-28. Molina I. (1992). Detection of proteolytic activity in microorganisms isolated from drycured ham. J. Food Sci., 57: 1308-1310.

280

F. NAZZARO et al.

Moller J.K.S., Hinrichsen L.L., Andersen H.J. (1998). Formation of amino acid (Lleucine, L-phenylalanine) derived volatile flavour compounds by Maroxella phenylpyruvica and Staphylococcus xylosus in cured meat model systems. Int. J. Food Microbiol., 42: 101-107. Molly K., Demeyer D., Johansson G., Raemaekers M., Ghistelinck M., Geenec I. (1997). The importance of meat enzymes in ripening and flavour generation of a European project. Food Chem., 59: 539-545. Montel M.C., Seronie M.P., Talon R., Hebraud M. (1995). Purification and characterization of a dipeptidase from Lactobacillus sake. Appl. Environ. Microbiol., 61(2): 837-839. Montel M.C., Reitz J., Talon R., Berdague J.L., Rousset A. (1996). Biochemical activities of Micrococcaceae and their effects on the aromatic profiles and odours of a dry sausage model. Food Microbiol., 13: 489-497. Roca M., Incze K. (1990). Fermented sausages. Food Rev. Int., 6: 91-118. Sarra P.G., Zacconi C., Scolari G. (2004). Characterization of specific microflora involved in "culatello" ripening. Ann. Microbiol., 54(1): 49-58. Selgas M.D., Sanz B., Ordonez J.A. (1986). Selection of micrococci strains for their use as a starter culture for dry-fermented sausages. In: The Proceedings of the 32nd European Meat Research Work. Gent, Belgium, pp. 251-257. Stahnke L.H. (1995a). Dried sausages fermented with Staphylococcus xylosus at different temperatures and with different ingredient levels. II. Volatile components. Meat Sci., 41: 193-209. Stahnke L.H. (1995b). Dried sausages fermented with Staphylococcus xylosus at different temperatures and with different ingredient levels. III. Sensory evaluation. Meat Sci., 41: 211-223. Stahnke L.H. (1999). Volatiles produced by Staphylococcus xylosus and Staphylococcus carnosus during growth in sausage minces - Part II. The influence of growth parameters. Lebensm-wiss. Techn., 32: 365-371. Talon R., Chastagnac C., Vergnais L., Montel M.C., Berdague J.L. (1998). Production of esters by Staphylococci. Int. J..Food Microbiol., 45: 143-150. Toldrà F., Rico E., Flores J. (1993). Cathepsin B, D, H and L activities in the processing of dry-cured ham. J. Sci. Food Agr., 62: 157-161. Toldrà F., Verplaetse A. (1995). Endogenus enzyme activity and quality for raw product processing. Composition of meat. In: Lundstrom, K., Hansson, I., Eds, Relation to Processing, Nutritional and Sensory Quality. Utrecht, The Netherlands, pp. 41-55. Verplaetse A., Gerard S., Buys E., Demeyer D. (1992). Endogenous and bacterial proteolysis in dry sausage fermentation. In: Proceedings International Congress Meat Science Technology, Clermont-Ferrand, France, pp. 851-854. Verplaetse A. (1994). Influence of raw meat properties and processing technology on aroma quality of raw fermented meat products. In: Proceedings of the 40th International Congress of Meat Science and Technology, The Hague, The Netherlands, pp. 45-65.

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