Effect of the addition of pancreatic lipase on the ripening of dry-fermented sausages — Part 1. Microbial, physico-chemical and lipolytic changes

ELSEVIER 0 3 0 9 - 1 7 4 0 ( 9 4 ) 0 0 0 3 1 -X

Meat Science 40 (1995) 159-170 Elsevier Science Limited Printed in Great Britain

0309-1740/95/$9.50

Effect of the Addition of Pancreatic Lipase on the Ripening of Dry-fermented Sausages - - Part 1.

Microbial, Physico-chemica| and Lipolytic Changes

M. Fernfindez, L. de la Hoz, O. Diaz, M. I. Cambero & J. A. Ord6fiez*

Departamento de Higiene y Tecnologia de los Alimentos, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

(Received 28 May 1994; revised version received 30 June 1994; accepted 29 July 1994)

ABSTRACT

The effect of the addition of nine different amounts (3-500 units) of pan- creatic lipase on the microbial and physico-chemical parameters and lipid fi'actions during the ripening of dry fermented sausages has been studied No d(fferences between conventional and iipase-added sausages were found for pH, dry matter and water activity. Tile addition of lipase caused a greater accumulation of pro&¢cts resulting from the triglyceride break- down, mainly diglycerides and fi'ee fittty acids ( FFA ). The maximum rate of lipolysis was observed during the first week o.]" the ripening process, specially in the fermentation phase. The greater the pancreatic lipase added, the higher lipolysis observed. At the end of the ripening, the levels of total FFA were clearly higher (I.5 to 5-fold) in all lipase.added batches than in the controls. This fact gave rise to the accumulation of a great amount of FFA, which can contribute either by themselves to the flavour of the sausage or t'an be available as substrates for further transformations which may generate other flavour compounds.

INTRODUCTION

The manufacture of dry fermented sausages is carried out in three main phases: formulation (including cutting and mixing of ingredients), fermentation and ripening. During the whole ripening process, several microbiological, physico- chemical and biochemical changes take place, which are responsible for the sen- sory quality and safety of this kind of product. In the feh'mentation, two basic

*Author to ~hom correspondence should be addressed.

159

160 M. Ferndndez et al.

microbial reactions occur simultaneously, i.e. the production of nitric oxide by nitrate- and nitrite-reducing bacteria (Micrococcaceae) and the decrease of the pH values via glycolisis by lactic acid bacteria (Liepe, 1982j. During the ripening phase, several other changes affect primarily proteins and fat and, through many secondary reactions, characteristic flavour compounds are formed (Liicke, 1984).

Fat constitutes the major fraction in these products and its degradation plays an essential role in dry sausage flavour (Demeyer et al., 1974). The first step in the lipid breakdown is the hydrolysis of triglycerides by both microbial (Papon et al., 1991) and endogenous lipases (Ferrer & Arboix, 1986) involving the liber- ation of fatty acids, which undergo later enzymic and non-enzymic oxidative processes, yielding, as final products, carbonyls and other small molecular weight compounds (alcohols, carboxylic acid, etc.), which are the main flavour com- pounds of the final product (Stahnke & Zeuthen, 1992).

The production of dry fermented sausages has a great tradition in Spain and many different varieties are made according to geographical regions. Dry fer- mented sausages manufactured in the traaitional way are actually being substi- tuted by industrial methods, trying to keep the original quality of the traditional ones. The production of dry fermented sausages in Spain represents about 170,000 tons, accounting for 18% of total meat products (M.A.P.A., 1992). Dry fermented sausages are also very popular among consumers in other countries of Continental Europe and America and now they are expanding throughout many countries where these products were hardly known some years ago, as Australia, Japan or Great Britain. The ripening of some sausage varieties can take several months. Thus, any attempt to accelerate the breakdown phenomena and to shorten the ripening process would be very useful for the meat industry. This acceleration would reduce the high cost of storage until the sausages reach a suitable matured state and, definitively, it would increase the profit margin and the competitivity of the product. Considering the importance of lipolytic phenom- ena during the ripening it seems interesting to explore the possibility of enhancing these reactions so that the product achieves the matured state in a shorter time than the usual one.

Several attempts have been made to accelerate the ripening of cheese, including ripening at high temperature, the use of modified starter cultures, the utiliza- tion of liquid slurries and the addition of enzymes (Law, 1984). Among them, enzyme addition has been the most studied. The acceleration of lipolysis by the addition of either animal or microbial lipases has been investigated in a lot of cheese varieties (Harboe, 1994). In particular, this procedure has been successfully applied to relatively strong-flavoured cheeses such as some Italian hard cheeses (Moskowitz, 1980) or blue cheeses (Jolly & Kosikowski, 1975), although some attempts on the use of lipases in other cheeses, as English Cheddar (Law & Wig- more, 1985) have failed. The addition of lipase has the combined effect of in- creasing the typical fatty acid flavour and catalysing a faster liberation of precursors of short-chain flavour compounds (Law, 1984). In general, polyunsat- urated fatty acids (PUFA) are oxidated faster when they are in their free form than when they are esterified to triglycerides or phospholipids (Cheftel & Chef- tel, 1980). Furthermore, in the lipolytic activity of dry sausages, lactic acid bacteria affects mainly short-chain fatty acid-triglycerides, although micrococci may also attack long-chain fatty acid-triglycerides (Selgas et al., 1986). According to this, the selective hydrolysis of fatty acids could be a relevant factor for lipid break-

Effect of pancreatic iipase on dry fermented sausages 161

down and flavour development. In the case of dry fermented sausages only few attempts have been made to accelerate the ripening process. In a previous work (Diaz et al., 1993), the effect of the addition of two different concentrations of pronase E on the proteolysis in dry fermented sausages was studied. Also, the effect of the addition of an animal (from throat glands of kid goat) (Paleari et al., 1991), and a microbial (from Lactobacillus plantarum) (Naes et al., 1992), lipase on the ripening of dry fermented sausages has been studied.

Because of this background, the presert investigation was undertaken. The goal was to study the effect of the addition of pancreatic lipase on the lipolysis in dry fermented sausages. Pancreatic lipase is a glycerol-ester hydrolase (EC 3.1.1.3). It was selected because this enzyme (Kristoffersen et al., 1967) and other similar animal lipases (Deeth & Fitz-Gerald, 1987; Fox & Guinee, 1987) have been frequently used in cheese manufacturing. Starting with a triglyceride, the relative rates of hydrolysis by this enzyme are triglycerides > diglycerides > monoglycerides. On the other hand, pancreatic lipase acts preferentially at the sn 1 and sn 3 positions of a triglyceride molecule (Whitaker, 1972).

MATERIALS AND METHODS

Materials

Five trials were made, in which nine batches of dry fermented sausages, with different amounts of added pancreatic lipase, were manufactured in an experi- mental plant of a local factory. The initial sausage mixture contained (% w/w): pork (57), beef (12), lard (25), NaC! (2.5), NaNO.~ (0.0095), NaNO2 (0.0065), sodium ascorbate (0.046), sodium glutamate (0.25), lactose (1.0), glucose (0.8), dextrine (1.25) and minced black pepper (0.14). Ingredients were mixed in a cutter with a particle size reduction of about 5 mm and were maintained at 8°C tbr 48 h. Then, difli:rent amounts (3, 10, 30, 40, 60, 90, 180, 250 and 500 units) of pan- creatic lipase (lipase crude type il, steapsin, fi'om porcine pancreas, Sigma Chemical Co., St. Louis, MO, U.S.A.) dissolved in 200 ml of distilled water were added to the sausage mixture. One lipase unit was defined as the amount of enzyme that yields 1 milliequivalent of fatty acid from olive oil in 1 h at 37°C and pH 7.7. One unit represented approximately 4 mg of the lipase extract per kg of the ini- tial sausage mixture. Each batch was named according to the number of lipase units added to the mixture. As a control, an additional batch was manufactured in each trial without adding the enzyme, but adding 200 ml of distilled water.

The mixture was stuffed into 40 mm diameter synthetic casings using a vac- uum filling machine. Sausages were ripened in a Kowell mod. CC3AFY cabinet (Montajes Navarra, Pamplona). Fermentation was carried out at 22°C and 90% relative humidity (RH) for the first 12 h and then the temperature and the RH were gradually reduced to 18°C and 80% RH in 60 h. Finally, sausages were dried at 12°C and 70-75% RH until the end of the ripening. The ripening period was 14 days for batches 10, 30, 40 and 60 (two trials), dried at 70% RH and 28 days for the remaining batches (3, 90, 180, 250 and 500) (three trials), dried at 75% RH. The controls were ripened in the same conditions as the corresponding batches. In all the figures, data from the controls are the mean values of the con- trol batches of the two trials of sausages ripened for 14 days (named as control

162 M. Ferndnde: et al.

14) and the three trials of sausages ripened for 28 days (named as control 28). Samples were taken at different times during ripening.

Microbial analyses

Microorganisms were counted by the pour-plate method using 1% peptone water as diluent. Total viable counts (TVC) were determined on Plate Count Agar (PCA) (Oxoid, Unipath Ltd., Basingstoke, Hampshire) and Micrococ- caceae on Manitol Salt Agar (MSA) (Oxoid), both incubated at 32°C for 2 days. Lactobaciili were enumerated on double-layer pH 5.6 MRS agar (Oxoid) after incubation at 32°C for 4 days.

Chemical analyses

A Crison Digit-501 pH-meter (Crison Instruments S.A., Barcelona) was used to measure the pH by inserting the electrode into the sausage sample. The measure- ment was made four times, changing the insertion place of the electrode. Dry matter was determined by drying at I I0°C to constant weight. Water activity (aw) was measured by duplicate at 25°C with a Decagon CXI dew point hygrometer (Decagon Devices Inc., Pullman, WA. U.S.A.).

Lipids were extracted and purified according to the method described by Han- son & Olley (1963). Total lipids were gravimetrically determined.

The separation of the different lipid classes was performed by thin layer chro- matograph~ (TLC) on 0.25 mm silica gel G-60 plates (Merck, Darmstadt) devel- oped with petroleum ether/diethyl ether/acetic acid (80/20/I) (v/v/v). Triolein, diolein, monolein, oleic acid and cholesterol (all from Sigma) were used as refer- ence standards. A spray of a 0.05% FeCI~.6H~O solution in a mixture of water/acetic acid/sultkiric acid (90/5/5)(v/v/v)(Lowry, 1968), lbllowed by heating in an oven at 120°C lbr 30 rain, was u.s'cd to visualize all lipid f"tactlons.-' " Lipid classes were quantified by densitometry in a Shimadzu CS-9000 densitometer (Shimadzu Corporation, K yoto) at 390 nm using calibration curves Ibr all the standards employed in TLC analysis.

RESULTS AND DISCUSSION

Microbial changes

The changes in the different microbial groups during ripening were similar in all control and iipase-added batches. Therefore, no efli:cts of the addition of pan- creatic lipase were observed. The microbial changes along ripening are exemplified by batch 180 and its control (Fig.l). The results of the remaining batches are not shown. The number of colonies developed on MSA stabilized at approximately 10~-I07 c.f.u./g. Lactobacilli increased rapidly during the first days to become stabilized afterwards at levels about 10x-10 '~ c.f.u./g. The counts on PCA and MRS were quite similar, which means that the dominant organisms along the ripening process were lactobacilli. This pattern was similar to that observed in conventional dry sausages (LOcke, 1984; Selgas et al., 1986; Garcia et al., 1992; Diaz et al., 1993).

Effect of pancreatic lipase on dry fermented sausages 163

9

0 4 8 t2 16 20 24 28

Time (days)

Fig. I. Changes in the microbial flora of lipase-added dry fermented sausages during the ripening process. Control batch (empty symbols); batch 180 (full symbols). Total viable count (squares); lactobacilli (circles); Micrococcaceae (triangles) All the control and the

nine lipase-added batches showed a similar pattern.

pH

'l'l~e initial pH (about 5.9 in all batches) dropped sharply during the first week of the ripening, specially in the fermentation phase (about 48-72 h). Thereafter, the values (4.9-5.1) became stabilized or increased slightly in the last stages of the process (Fig. 2). This increase may be explained by the accumulation of basic nitrogen compounds as a restllt oi" the protein breakdown (Ltlcke, 1984: Vet'- phlelse el al., 1989). No important dil'ferenccs were observed between control and iipase-added batches, e×cepling Ibr batches 250 and 500, in which the final

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Fig. 2. Changes in the pH of lipase-added dry fermented sausages during the ripening pro- cess. CA) 14 days of ripening: (El) control 14 (average of two batches); CA) batch 10: (A) batch 30; (O) batch 40; (O) batch 60. (B) 28 days of :'ipenmg: ([]) control 28 (average of

three batches): ( I ) batch 3; CA) batch 90; (,A) batch 180: (O) batch 250: (O) batch 500.

164 M. Fern4ndez et ai.

values were higher, probably due to the proteolytic activity present as impurity in the lipase crude extract (as reported in the label by the manufacturer). The pH values determined in the present work were similar to those reported for different American (Acton & Dick, 1976; Palumbo et al., 1976) and European (Sanz et al., 1988; Stiebing & ROdel, 1990; Garcia et aL, 1992; Samelis et al., 1993) dry or semidry fermented sausages.

Water content and water activity

The water content after formulation represented about 60% of the fresh product in all batches and decreased during the ripening process to reach 40-45% of the final product (Fig. 3). These final values were only slightly higher in the sausages ripened for 14 days, because, as observed by other authors (Incze, 1987; Stiebing & R0del, 1987), the deeper rate of dehydration occurs during the second stage of the process. These results are amid the range expected for dry fermented sausages (Lticke, 1984; Nychas & Arkoudelos, 1990). As a result of the water loss, the final weight loss was about 15-20%, common of semidry sausages (Lticke, 1984).

Water activity (aw) of the initial mixture was 0.97, decreasing gradually during ripening to reach values of 0.90-0.92 in batches ripened for 14 days and 0.86-0.87 in those ripened for 28 days (Fig. 4). No important effects of the addi- tion of pancreatic lipase were observed. These results coincide with those reported in the most dry fermented sausages (Palumbo et al., 1976; LOcke, 1984; Sanz et a1.,1988).

L|pid fractions

The total tilt content (Fig. 5) increased along ripening according to water loss (Fig. 3). The initial fitt content was about 23% (wet weight) increasing gradually throughout the ripening. The linal product contained 35+39'¼. tat (5963% of dry

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Fig. 3. Changes in the water content (%) of lipase-added dry fermented sausages during the ripening process. (A) Fourteen days of ripening: (El) control 14 (average of two batches); (A) batch 10; (,A) batch 30; (O) batch 40; (O) batch 60. (B) Twenty-eight days of ripening: (~) control 28 (average of three batches); (M) batch 3; (A) batch 90; (A)

batch 180; (O) batch 250; (0) batch 500.

Effect of pancreatic lipase on dry fermented sausages 165

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Fig. 4. Changes in the water activity (aw) of lipase-added dry fermented sausages during the ripening process. (A) Fourteen days of ripening: (I-l) control 14 (average of two batches (A) batch 10: (A) batch 30; (O) batch 40; (O) batch 60. (B) Twenty-eight days of ripening: (I-I) control 28 (average of three batches); ( I ) batch 3; (A) batch 90; (A) batch

180; (O) batch 250; (0) batch 500.

matter). These values are normal for conventional dry sausages (Acton & Dick, 1976; Ferrer & Arboix, 1986; Samelis et al., 1993). In batches 250 and 500 (Fig. 5B) the lipid content was clearly lower than that of the other batches, about 30% in the final wet product and 50% in terms of dry matter (D.M.). It is not clear why the determination of total fat yielded low amounts in those batches, but it may be explained by a probable loss of polar glycerides (probably monoglycerides) resulting from the high lipolytic activity observed in these batches, which could remain retained ill the aqueous phase during the fat extraction process.

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Fig. 5. Changes in the fat content (% of wet weight) of lipase-added dry fermented sausages during the ripening process• (A) Fourteen clays of ripening: (0) control 14 (average of two batches); (A) batch 10; (&) batch 30; (O) batch 40; (0) batch 60. (B) Twenty-eight days of ripening: (U]) control 28 (average of three batches); (RI) batch 3;

(A) batch 90; (&) batch 180; (O) batch 22;0: {~) b~1ch 500.

166 M. Fern4nde'. et al.

TLC analysis revealed the presence of eight to nine spots in the lipid extracts from all batches. According to their Rrs and the behaviour against general and specific reagents, six of them were characterized as monoglycerides (Rr = 0.01), diglycerides (Rf = 0.12), free cholesterol ( R f = 0.17), free fatty acids (FFA) (Rf = 0.26), triglycerides (Rf = 0.80) and hydrocarbons plus cholesteryl esters (Rr = 0.96). These substances are the same as those reported in the meat fat from sev- eral animals (Chang-Han & Yeon-Hee, 1982; Cambero et al., 1991). Two or three spots with Rfs close to that of FFA were not ~'dentified.

For monitoring the lipolytic changes in the experimental sausages, triglyc- erides, diglycerides, monoglycerides and FFA were quantified. In the TLC sys- tem used in this work, the mobility of monoglycerides was very low (Rr = 0.01) and, therefore, they remained together with phospholipids at the origin of the TLC plates. For this reason, both types of compounds were quantified together. However, phospholipids show only slight changes during the ripening process (Demeyer et al., 1974). Thus, the increases observed in this spot were attributed to the accumulation of monoglycerides.

The triglyceride content was 44-47 g/100 g D.M. (about 80-90% of total lipids) in the initial mixture and decreased up to 41-43 g/100 g D.M. (6.8-8.5%) in control sausages at the end of the ripening process (Fig. 6). In lipase-added batches, the greater the pancreatic lipase content the higher the decrease of triglycerides, losing up to two-thirds of the initial content in batch 500 (Fig. 6). The monoglyceride content increased from 2-3 g/100 g D.M. at the beginning of the ripening to about 4 g/100 g D.M. in the control batches (Fig. 7). The final content of monoglycerides in lipase-added batches was slightly higher than that of the controls in batches added with less than 60 lipase units, but these com- pounds reached values 2-3-fold higher in batches manuthctured with 60 or more lipase units. Because of the defective fat extraction in batches 250 and 500 (Fig. 5B), data of monoglyceride content from these batches are not shown in Fig. 7. Diglycerides were initially detected in trace amounts (less than 0.03 g/100 g

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Fig. 6. Changes in the triglyceride content (% of dry matter) of lipase-added dry fermented sausages during the ripening process. (A) Fourteen days of ripening: (1[]) con- trol 14 (average of two batches); (A) batch 10; (&) batch 30; (O) batch 40; (0) batch 60. (B) Twenty-eight days of ripening: (I-1) control 28 (average of three batches); ([]) batch 3;

(A) batch 90; (&) batch 180; (O) batch 250; (O) batch 500.

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Effect of pancreatic lipase on dry fermented sausages 167

Fig. 7. Changes in the monoglyceride content (% of dry matter) of lipase-added dry fermented sausages during the ripening process. (A) Fourteen days of ripening: (I-q) con- trol 14 (a~erage of two batches); (A) batch 10; (A) batch 30; (C)) batch 40; (O) batch 60. (B) Twenty-eight days of ripening: (I-q) control 28 (average of three batches); ( I ) batch 3;

(A) batch 90; (A) batch 180.

D.M.), but clearly increased during ripening to reach from 1.9 g/100 g D.M. in batch 3 to approximately 15 g/100 g D.M. in batches added with more than 180 lipase units (about 2--4-fold higher than those values determined in control sausages) (Fig. 8). The initial FFA content was similar to diglycerides and increased from 3 g/100 g D.M. (batch 3) to 9 g/100 g D.M. (batch 500) which means an increase of 1.5-5-fold in relation to the controls (Fig. 9). The high final content of FFA detected in batches 90, 180, 250 and 500 (about 7-9 g/100 g D.M.) has only been described in conventional sausages with a long

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Fig. 8. Changes in the diglyceride content (% of dry matter) of lipase-added dry fermented sausages during the ripening process. (A) Fourteen days of ripening: (I-3) con- trol 14 (average of two batches); (A) batch 10; (A) batch 30; (O) batch 40; (0) batch 60. (B) Twenty-eight days of ripening: (7-]) control 28 (average of three batches); (R) batch 3;

(A) batch 90; (A) batch 180; (O) batch 250; (O) batch 500.

168 M. Ferminde: et al.

(more than 3 months) ripening period (Ferrer & Arboix, 1986; Lois et al., 1987).

In short, a continuous decrease in the triglyceride content of all sausages took place during the ripening, which, in turn, produced increases in diglyceride, FFA and, less prominently, monoglyceride fractions. The lipid breakdown at the end of the ripening is summarized in Table 1, in which the percentage of decrease of triglycerides and the percentage of increase of monoglycerides, diglycerides and FFA of all the lipase-added batches, related to the final values of the controls, are shown. In general, the higher the amount of lipase added, the greater the lipolysis observed. The results obtained in control batches were similar to those reported for different dry or semi-dry sausages (Demeyer et al., 1974; Le6n Crespo et al., 1985; Paleari Bianchi et al., 1985; Dominguez & Zumalacfirregui, 1991). As it can be observed in Figs 6-9, the lipolytic changes were greater in all lipase- added batches (mainly in those manufactured with more than 60 lipase units) than those of the control sausages. The greater lipid breakdown was observed during the fermentation phase, followed by a lower rate of lipolysis afterwards. The higher accumulation of products resulting from lipolysis in all lipase-added batches during the fermentation phase has been attributed to the pH (about 6.0) and the temperature (22°C) of the sausages during this per iod--not far from the optimum conditions of the pancreatic lipase activity (pH 7.0 and 37°C). Higher contents of monoglycerides, diglycerides and FFA than those determined in sausages added with 180, 250 and 500 units of pancreatic lipase could be expected. However, the pattern observed in these batches was quite similar to that found in batch 90. This fact may be due to a retroinhibition of the enzyme caused by the great accumulation of the reaction products, mainly FFA (Drapon, 1972; Ferreira & Patton, 1990). The lower rate of lipolysis observed after the fermentation phase may be considered as a desirable circumstance, because, in this way, an excessive lipid breakdown and the corresponding over- maturation of the product during the commercial lil'e may be avoided.

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Fig. 9. Changes in the total free fatty acid content (% of dry matter) of lipase-added dry fermented sausages during the ripening process. (A) Fourteen days of ripening: (D) con- trol 14 (average of two batches); (A) batch 10; (&) batch 30; (O) batch 40; (0) batch 60. (B) Twenty-eight days of ripening: ([]) control 28 (average of three batches): ([i) batch 3;

(A) batch 90; (&) batch 180; (O) batch 250; (0) batch 500.

Effect o f pancreatic iipase on dry fermented sausages 169

TABLE 1 Decrease (%) of the Triglyceride Content and Increase (%) of the Diglyceride, Mono- glyceride and Total FFA Contents in Dry Fermented Sausages with Different Amounts of Pancreatic Lipase Added at the End of the Ripening in Relation to the Control Batches

Lipase Batch units TG MG DG FFA

3(2) 3 0.5 11.8 74.9 54.2 10(1) 10 0.1 2.4 89.1 45.1 30(1) 30 0-2 2- I 133.0 106.4 40(1) 40 1.5 14.7 282.8 263.7 60(1) 60 6.6 51.2 166.4 190.2 90(2) 90 16.4 76.7 760.3 255.7

180(2) 180 35.9 55.4 1230.5 324.3 250(2) 250 58.2 ND 1370.9 325.5 500(2) 500 66. i ND 1324.8 351.7

TG, triglycerides; MG, monoglycerides; DG, diglycerides; FFA, total free fatty acids; ND, not determined; (1) batches ripened for 14 days; (2) batches ripened for 28 days.

In conclusion, a strong acceleration of the lipolytic phenomena during the ripening of dry fermented sausages has been achieved by the addition of pan- creatic lipase. The resulting products (mainly FFA) may contribute either by themselves to the flavour of the sausage or be available as substrates for further transformations, which may generate other flavour compounds. These effects are reported in the subsequent paper (Fern~indez et aL, 1995).

ACKNOWLEDGEMENTS

This work was supported by the Comisi6n lnterministerial de Ciencia y Tec- nologia (CICYT, Spain), project AL, I 88/0005. Manuela Fermindez and Olga Diaz were beneficiaries of grants of Formaci6n de Personal Investigador from the Comunidad Aut6noma de Madrid and the Ministerio de Educaci6n y Cien- cia, respectively. We thank lndustrias CABO for graciously providing the ingre- dients and for manufacturing the experimental sausages.

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