Meat Science 66 (2004) 415–423

www.elsevier.com/locate/meatsci

Lipolysis and lipid oxidation in fermented sausages depending on

different processing conditions and different antioxidants
Emanuela Zanardi*, Sergio Ghidini, Alessandra Battaglia, Roberto Chizzolini
Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualita’ e Sicurezza degli Alimenti, Università degli Studi di Parma,
via del Taglio 8, 43100 Parma, Italy

Received 1 October 2002; received in revised form 28 April 2003; accepted 20 May 2003

Abstract
Lipolysis and lipid oxidation in Mediterranean and North Europe type sausages were studied in relation to raw material, pro-
cessing conditions and additives. In particular the effect of ascorbic acid, nitrites and spices was evaluated. Lipolysis was measured
by the determination of total and free fatty acids of fresh minces and matured products and lipid oxidation was evaluated by
thiobarbituric acid reactive substances and cholesterol oxidation products. The increase of free fatty acids during maturation
appears to be independent from processing conditions and the differences in polyunsaturated fatty acids increment found among
the formulations appear to be due to inherent variations of raw materials. The presence of ascorbic acid and/or nitrite seems
important for cholesterol protection and, as a consequence, for the safety of fermented meat products while spices at doses up to
0.1% do not seem to have a remarkable effect. The effect on fatty acid oxidation of the same additives and of the different pro-
cessing technologies is not significantly different and the variations linked to raw material may play the greatest role.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Fermented sausage; Lipolysis; Lipid oxidation; Antioxidants

1. Introduction oxidation by addition of lipases (Ansorena, Zapelena,

Lipids in dry cured meat products are hydrolysed by
lipases with production of free fatty acids (Coutron-
Gambotti & Gandemer, 1999; Gandemer, 2002). Such
lipases are mainly of endogenous origin (Molly, et al.,
1997) and preferentially release linoleic and oleic acids
(Demeyer, Hoozee, & Mesdom, 1974). This is due both
to the specificity of lipases for the position sn-3 of tri-
glycerides and to the activity of phospholipases on phos-
pholipids (Molly, Demeyer, Civera, & Verplaetse, 1996).
The latter compounds, because of their high content of
long chain polyunsaturated fatty acids, indeed contribute
significantly to the increase of free unsaturated fatty acids
during sausage maturation (Alasnier, & Gandemer, 1998).
Free fatty acids are more susceptible to oxidation than
their counterparts bound to triglycerides and phospholi-
pids, as demonstrated by the decrease of their relative
amounts in the final phases of maturation (Coutron-
Gambotti & Gandemer, 1999) and the increase of lipid
* Corresponding author. Tel.: +39-0521-032761; fax: +39-0521-
032752.
E-mail address: emanuela.zanardi@unipr.it (E. Zanardi).
0309-1740/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0309-1740(03)00129-3
Astiasarán, & Bello, 1998). Other work however has
shown that increased lipolysis was not associated with
increased rancidity (Fernandez & Rodriguez, 1991; Gan-
demer, 2002; Nagy, Mihályi, & Incze, 1989).
Fermented meat products are rich in lipids and, in the
case of Milano sausages for instance, a significant per-
centage of them is made up by monounsaturated (about
45%) and polyunsaturated fatty acids (about 13%)
(Zanardi, Dorigoni, Badiani, & Chizzolini, 2002). Oxi-
dation of unsaturated fatty acids can occur during
maturation and, to a certain extent, it is a useful phe-
nomenon as it produces compounds active on the front
of flavour (e.g. aroma and taste) (Ordoñez, Hierro,
Bruna, & de la Hoz, 1999). An excess of oxidation,
though, could reach the level of rancidity, and the
foodstuff would no longer be acceptable for human
consumption, but, before such a condition is reached,
lipid oxidation could generate toxic molecules with
possible hazards for human health.
Oxidative stress is, indeed, considered to be respon-
sible for a variety of very damaging chronic degen-
erative diseases to the point that the knowledge of the
interactions between oxidants and antioxidants in
416 E. Zanardi et al. / Meat Science 66 (2004) 415–423
human health has been defined as important as the dis-
covery of the role of antibiotics and microrganisms in
infectious disease (Bray, 2000). Nutrition, in such a
context, occupies an extremely relevant role as food is a
primary source of antioxidants but it can also present
the risk of carrying active oxygen species, and some
lipid oxidation products are among the latter.
Production technology of dry fermented sausages
makes use of substances with pro-oxidant and anti-oxi-
dant activity. Sodium chloride, the prime added com-
pound, has prooxidant effects and nitrates/nitrites are
involved in oxidation-reduction reactions in meat pro-
ducts as clearly exemplified by the chemical pathways
known to lead to the formation of nitrosylmyoglobin in
cured products (Freybler et al., 1993; Skibsted, 1992).
Sodium nitrite, per se a strong oxidant, in cured meats
has antioxidant activity through the production of
nitric oxide (Kanner, 1994; Skibsted, 1992) and a
number of investigations have reported its positive
effects in controlling rancidity and off flavours devel-
opment in cured meat products (Freybler et al., 1993;
Igene, Pearson, Merkel, & Coleman, 1979; Morrissey &
Tichivangana, 1985). Other compounds and/or sub-
stances are used to reduce oxidative processes or are
credited with antioxidant activity (ascorbates, spices,
phosphates, etc.).
The aim of the research that has been carried out was
to investigate the possible relationship between lipolysis,
oxidation and antioxidant additives in the production of
typical dry cured fermented sausages of different Eur-
opean origin. European fermented meat products can
be roughly classified as Mediterranean, or Southern
Europe type, generally characterised by long maturing
times, slow pH fall, pH of matured products higher than
5, flavour significantly affected by the use of spices, and
Northern Europe type, characterised by fast pH fall, final
pH lower than 5, smoking and shorter maturing times.
The investigation was based on two southern and two
northern type fermented meat products: one southern
(M1) produced by an industrial firm located in a south
European country, a second southern sausage (M2)
from a producer of north Europe who also produced a
northern type product (N1) and a second northern type
sausage (N2) from a third firm of north Europe. The
results described were obtained within the framework of
a European project (Demeyer, 2001).
2. Materials and methods
The first part of the research was directed at compar-
ing four types of fermented sausages produced accord-
ing to standard formulations whereas in the second part
of the work the formulation of one of the southern type
sausages was modified to study the role played by spi-
ces, nitrates/nitrites and ascorbate.
Five batches of each type have been produced for the
first phase with the following formulations.
2.1. Mediterranean type sausage M1
Fresh pork shoulders (72%) and pork streaky bacon
(28%) were minced to 3.5 mm particle size and mixed
with salt (2.5%), skimmed milk (2.8%), NaNO2 (80
ppm), KNO3 (120 ppm), sucrose (0.5%), dextrose
(0.3%), ascorbic acid (0.03%), black pepper (0.07%),
white pepper (0.03%), garlic (0.01%). A commercial
mixture of Lactobacillus curvatus and Micrococcus
varians was used as starter culture. Processing tempera-
ture went from 21.5  C of fermentation to 12.5  C of
maturation. Total processing lasted 40 days.
2.2. Mediterranean type sausage M2
Fresh (33%) and frozen (33%) lean pork cuts and
pork back fat (33%) were minced to 2.5 mm particle
size and mixed with salt (3%), caseinate (1.15%),
NaNO2 (180 ppm), dextrose (0.5%), sodium ascorbate
(0.09%) and black pepper (0.07%). A mixture of Lacto-
bacillus sake, Pediococcus pentosaceus, Staphylococcus
xylosus and M. varians was used as starter culture. Pro-
cessing temperature went from 24.5  C of fermentation to
12.5  C of maturation. Total processing time was 28 days.
2.3. Northern type sausage N1
Frozen lean beef (33%), fresh pork (33%) and pork
back fat (33%) were minced to 2.5 mm particle size and
mixed with salt (3%), caseinate (1.15%), NaNO2 (180
ppm), dextrose (0.7%), sodium ascorbate (0.09%),
black pepper (0.07%). A mixture of L. sake, P. pento-
saceus, St. xylosus, Staphylococcus carnosus and M.
varians was used as starter culture. Processing tempera-
ture went from 26  C of fermentation to 20  C of
maturation. The sausages were smoked for 1 h and total
processing time took 14 days.
2.4. Northern type sausage N2
Frozen and fresh beef (30%), frozen pork (40%) and
pork back fat (30%) were minced to 2.0 mm particle
size and mixed with salt (4%), NaNO2 (240 ppm), dex-
trose (0.43%), a mixture of oleoresins (mainly black
pepper with coriander, paprika and cognac- 0.014%)
and garlic (0.04%). A mixture of Lactobacillus (strain
not determined) and St. carnosus was used as starter
culture. Processing temperature went from 27  C of fer-
mentation to 14  C of maturation. The sausages were
smoked for 2.5 h and total processing time was 21 days.
Two batches of three different formulations were
produced for the second part of the study using the M1
type as control. The formulation of M1 was modified in
 E. Zanardi et al. / Meat Science 66 (2004) 415–423 417

one case (M1–F1) to include only 150 ppm of NaNO2
(without KNO3, ascorbic acid and spices), in the second
case (M1–F2) to include only ascorbic acid 0.03%
(without nitrate/nitrite and spices) and in the third case
(M1–F3) to include only spices 0.11% (without nitrate/
nitrite and ascorbic acid).
Proximate composition, NaCl, pH, non protein
nitrogen, residual nitrites and nitrates, fatty acid com-
position (Zanardi, Novelli, Ghiretti, & Chizzolini,
2000), free fatty acid composition (Garcia Regueiro,
Gilbert, & Diaz, 1994), TBARS (Novelli et al., 1998),
total cholesterol and cholesterol oxides (Zanardi et al.,
1998) were determined at the end of processing.
by the measurement of non protein nitrogen (nitrogen
containing fractions that are not precipitated by 10%
trichloracetic acid) that remained constant on values
around 13–14% irrespective of the technology adopted
(raw materials, mincing level, starter cultures, additives,
processing times and conditions).
The overall result is in line with the general conclusion
that proteolysis, down to free amino acids is mainly the
result of muscle enzymes and not much influenced by
starter cultures, as also concluded from studies using an
aseptic meat model system (Claeys, De Smet, Demeyer,
Geers, & Buys, 2001; Harnie, Claeys, Raemaekers, &
Demeyer, 2000).

3.2. Free fatty acids and lipolysis

3. Results and discussion
Total fatty acid composition in minces is collected in
3.1. Compositional data and proteolysis Table 2. No significant differences between minces and

Sausages belonging to the M1 formulation had aver- Table 2

age moisture contents ranging from 38 to 39% Fatty acid composition (% of total extractable lipids) of M1, M2, N1
approximately, fat from 31 to 33%, protein from 23.6 to and N2 minces (mean valuesstandard deviation)a
24.2%, NaCl from 4 to 4.3% and NPN from 13.7 to Formulations
14.5% (Table 1). The formulation named M1 differed
from M2 with a higher fat content, which affected M1 M2 N1 N2
moisture and protein contents. The formulation N1 had C8:0 0.02 0.01 0.01 0.01 0.01 0.01 0.010.01
the highest moisture content of the four formulations, C10:0 0.11 0.01 0.05 0.01 0.05 0.01 0.070.01
coupled with the lowest protein and NaCl contents and, C12:0 0.13 0.01 0.16 0.02 0.16 0.03 0.130.01
C14:0 1.72 0.01 1.32 0.05 1.36 0.12 1.420.04
also, the lowest pH value. The forth formulation (N2)
C14:1 0.03 0.01 0.03 0.01 0.04 0.01 0.070.02
was similar to the third one (N1) for most parameters C16:0 25.5 0.27 22.6 0.25 22.7 0.56 25.50.27
except for the higher NaCl content. The last three for- C16:1 2.39 0.01 2.66 0.16 2.56 0.12 3.030.04
mulations had a composition similar to M1 except for C18:0 12.4 0.04 13.5 0.24 14.0 1.00 16.31.19
pH values that are between 0.4 and 0.7 units higher in C18:1 42.8 0.36 41.2 0.31 40.9 0.70 40.10.88
C18:2 12.5 0.54 15.2 0.30 14.9 1.07 10.70.32
M1–F1, M1–F2 and M1–F3. Fat content on a dry
C18:3 0.50 0.03 0.96 0.05 1.02 0.06 0.690.04
weight basis was around 50% or lower in M1, M1–F1, C20:0 0.17 0.01 0.15 0.01 0.16 0.01 0.180.01
M1–F2 and M1–F3 and around 60% in the other C20:1 0.84 0.02 1.15 0.02 1.13 0.10 1.050.03
formulations. C20:2 0.57 0.04 0.77 0.03 0.73 0.04 0.550.01
Compositional data, besides confirming the known C20:4 0.30 0.04 0.34 0.01 0.32 0.03 0.270.04
pH difference between south and north European sau- Saturated 40.1 0.31b 37.7 0.16b 38.4 1.58b 43.61.16a
sages, have shown some technologically significant Mono 46.1 0.34a 45.1 0.45a 44.7 0.72a 44.20.90a
divergences in fat, protein and NaCl contents. The Poly 13.9 0.66b 17.2 0.36a 16.9 1.17a 12.20.38b
above mentioned differences, though, did not seem to a
Different letters, in a line, stand for significant differences,
have significant effects on gross proteolysis, as evaluated P40.05, Scheffè’s test.

Table 1
Proximate composition (%), NaCl content (%), non protein nitrogen (NPN,% of total nitrogen) and pH in matured sausages (mean values
standard deviation)

Formulations

M1 M2 N1 N2 M1–F1 M1–F2 M1–F3

Moisture 38.60.6 30.01.0 42.80.9 41.11.1 41.21.2 42.82.0 41.20.1

Fat 31.90.8 42.80.9 35.70.9 34.01.4 29.50.5 25.21.4 28.50.3
Protein 24.00.2 22.01.0 17.60.6 18.80.5 23.30.3 25.90.7 23.70.1
NaCl 4.20.1 4.30.1 3.20.1 4.70.1 4.20.1 4.10.2 4.10.2
NPN 14.20.3 13.31.3 13.01.6 13.51.6 13.70.4 12.70.1 14.30.1
pH 5.550.06 5.790.33 4.500.06 4.680.08 6.210.01 6.010.33 5.920.22
418 E. Zanardi et al. / Meat Science 66 (2004) 415–423
matured products were observed in the total fatty acid
composition. The four types of products, as can be
easily observed from the distribution of the three main
groups (saturated, monounsaturated and poly-
unsaturated) do not differ appreciably, apart from the
N2 formulation. The quota of polyunsaturated fatty
acids of the latter is about 4–5 points lower compared to
formulations N1 and M2, to the advantage of saturated
acids, probably due to the presence of a certain amount of
beef fat. Both in minces and matured sausages the main
acids of formulations M1, M2 and N1 were palmitic (23–
26%), stearic (12–15%), oleic (41–42%) and linoleic (13–
15%); total saturated fatty acids were about 37–41%,
monounsaturated around 44-46% and polyunsaturated
between 14 and 17%. No significant differences were
observed between M1 on one side and M1–F1, M1–F2,
M1–F3 formulations on the other (data not shown).
The distribution of free fatty acids (FFA) did not
follow the same pattern just reported for total fatty
acids (Table 3). The minces of the four formulations
were characterised by a higher proportion of saturated
fatty acids (48.4%) in M1 compared to M2 (40.8%), N1
(41.3%) and N2 (45.4%) whereas no significant differ-
ences were observed for monounsaturated (between 35
and 41%) and polyunsaturated (between 16 and 20%)
fatty acids. Free fatty acid compositions of matured
sausages differed among the four formulations: in for-
mulation N2 the polyunsaturated are around 23%, sig-
nificantly lower compared to M1, M2 and N1, and the
saturated are 34.6%, significantly higher than the other
three formulations. Significantly lower values are
reported in Demeyer (2001), probably because of erro-
neous separation of the free fatty acid fraction in the
laboratory concerned (Demeyer, personal communi-
cation). The highest amount of monounsaturated
(44.6%) was recorded in formulation M2 whereas the
amount in the other three formulations scored between
40.3 and 42.5%. Formulations M2 and N1 were very
similar, reflecting their origin from the same producer
Table 3
Free fatty acid content (%) of M1, M2, N1 and N2 fresh minces and matured sausages (mean valuestandard deviation)a
M1
Minces
SATURATED 48.41.17a
MONO 35.11.05a
POLY 16.40.16a
FFA (% of total fatty acids) 1.450.12
Matured products
SATURATED 33.10.03ab
MONO 40.32.30b
POLY 26.72.31a
FFA (% of total fatty acids) 4.520.36
a
Different letters, in a line, stand for significant differences, P40.05, Scheffè’s test.
and, therefore, the same raw materials apart from the
presence/absence of beef lean (the fat content of which
was found to be < 5%). The reasons for such differences
are difficult to imagine apart from the possible effect of
raw materials coming from different pig genetic lines
and feeding practices.
Maturation brought about a widespread increase in
FFA that went from less or a little more than 1% of
total fatty acids in the minces to a minimum of 2.17% in
M2 and a maximum of 4.52% in M1 (Table 3). All main
fatty acids increased to a significant extent but the phe-
nomenon was more marked for the unsaturated ones.
Free monounsaturated fatty acids increased their inci-
dence of about 5 percentage points in all formulations
except N1 in which they raised only from 39.1 to 41.5%.
The relative and absolute increments of polyunsaturated
fatty acids were higher than those of monounsaturated
ones with the result that their incidence exceeded 22%
in all formulations. The highest value was observed in
M1 (26.7%), close to M2 (24.1%) and N1 (26.4%) but
significantly above the N2 (22.9%) (Table 3).
The amount of free fatty acid was between 7 and 10
mg/g fat in minces and between 25 and 32 mg/g fat in
matured products (Table 4). Free fatty acid changes
following maturation are more evident through the
ratios between the values of the minces and those of
matured products. The highest relative increase was
scored by N1 (4.2) but the value was not significantly
different from those of other formulations that ranged
from 2.6 of M2 to 3.1 of M1. No significant differences
among the formulations have been observed in relative
increases of saturated and monounsaturated free fatty
acids. The general trend was of a rise by a factor of two,
approximately, for saturated fatty acids and by a factor
of about 3–3.5 for monounsaturated acids. The increase
of polyunsaturated was by a factor of about 5 in M1
and N1 and of 3.4–4.1 in M2 and N2 (Table 4).
Free fatty acid content of formulations M1–F1, M1–
F2 and M1–F3 followed the lead of M1, as for other
Formulations
M2 N1 N2
40.8 1.97c 41.32.44bc 45.4 2.51ab
40.6 2.79a 39.14.50a 37.3 2.08a
18.5 1.14a 19.73.88a 17.2 2.50a
1.01 0.26 0.650.11 0.99 0.32
31.3 1.17b 32.10.90b 34.6 1.41a
44.6 1.29a 41.51.51b 42.5 1.35ab
24.1 0.94ab 26.41.90a 22.9 0.78b
2.17 0.29 2.590.23 2.75 0.24
 E. Zanardi et al. / Meat Science 66 (2004) 415–423 419

Table 4
Free fatty acids content (mg/g fat) of M1, M2, N1 and N2 type sausages: fresh mince and matured product (mean valuesstandard deviation)a

Formulations

M1 M2 N1 N2

Mince Matured Mince Matured Mince Matured Mince Matured

C10:0 0.160.01 0.230.07 0.12 0.02 0.15 0.04 0.100.02 0.110.01 0.13 0.07 0.140.03
C12:0 0.060.01 0.190.07 0.09 0.03 0.10 0.02 0.050.03 0.100.02 0.13 0.07 0.120.03
C14:0 0.240.01 0.690.06 0.22 0.05 0.48 0.11 0.180.04 0.580.09 0.25 0.10 0.730.25
C16:0 2.810.06 6.160.82 2.19 0.56 4.48 0.66 1.530.47 5.530.78 2.31 0.83 5.870.73
C16:1 0.220.02 0.750.13 0.24 0.06 0.60 0.09 0.180.06 0.620.12 0.28 0.09 0.940.13
C18:0 1.630.01 3.210.26 1.47 0.28 2.60 0.25 1.210.21 3.190.47 1.82 0.45 3.420.27
C18:1 3.240.11 11.62.00 3.87 1.04 10.2 1.01 2.750.82 10.81.77 3.53 1.04 11.21.00
C18:2 1.350.04 6.470.53 1.47 0.31 4.88 0.52 1.020.17 5.431.17 1.21 0.28 5.310.53
C18:3 0.080.02 0.360.12 0.16 0.03 0.48 0.05 0.130.04 0.580.11 0.14 0.06 0.610.06
C20:1 0.080.03 0.480.02 n.d. 0.33 0.08 n.d. 0.320.07 n.d. 0.410.10
C20:2 0.120.01 0.790.16 0.23 0.19 0.33 0.06 0.260.14 1.190.29 0.36 0.18 0.420.03
C20:4 0.110.01 0.800.30 n.d. 0.31 0.05 n.d. 0.270.18 n.d. 0.440.06
Total FFA 10.10.14 31.73.43 10.1 2.32 25.0 2.68 7.411.54 28.24.60 10.2 2.78 29.62.73
Saturated FFA 4.890.05 10.51.15 4.09 0.88 7.82 1.04 3.070.69 9.021.27 4.65 1.39 10.31.21
Mono FFA 3.550.16 12.82.11 4.11 1.09 11.1 1.16 2.930.87 11.71.94 3.81 1.10 12.61.11
Poly FFA 1.660.04 8.400.18 1.85 0.40 6.00 0.58 1.410.12 7.471.54 1.72 0.38 6.790.59
Total FFA matured/ 3.10.4a 2.60.6a
total FFA mince 4.21.5a 3.0 0.7a
Sat. FFA matured/ 2.10.2a 2.00.5a
sat. FFA mince 3.31.2a 2.3 0.6a
Mono FFA matured/ 3.60.8a 2.90.8a
mono FFA mince 4.71.8a 3.5 0.8a
Poly FFA matured/ 5.10.2ab 3.40.7b
poly FFA mince 5.41.4a 4.1 0.8ab
a
Different letters, in a line, stand for significant differences, P40.05, Scheffè’s test; n.d.: not detectable.

parameters, with the only divergence of M1–F3 in
which the nearly 13 mg/g fat of free fatty acids observed
in the mince, the highest among the seven formulations,
determined a significant lower ratio between free fatty
acids in matured products and minces: the value was 2.3 in
M1–F3 and around 3 for the other formulations (Table 5).
The observed changes in FFA are in line with results
reported by other investigators. The specific liberation of
polyunsaturated fatty acids, and of monounsaturated
ones to a lower extent, had been reported by Demeyer et
al. (1974) and confirmed by several studies, especially in
subcutaneous adipose tissue of dry cured hams (Ante-
quera, Córdoba, Ruiz, Martı́n, Bermúdez, & Ventanas,
1993; Coutron-Gambotti & Gandemer, 1999; Martı́n,
Córdoba, Ventanas, & Antequera, 1998; Vestergaard,
Schivazappa, & Virgili, 2000). Lipases attack preferentially
the fatty acids placed on the outer position of the trigly-
cerides molecules, and position 3 more than position 1,
where unsaturated fatty acids are predominantly placed.
Domı́nguez Fernández and Zumalacárregui Rodrı́-
guez (1991) observed a marked increase in FFA during
the maturation of chorizo prepared in four different
formulations regarding ingredients, salt, additives, spi-
ces, with and without nitrates and nitrites. Total FFA of
such chorizos at the end of maturation varied
considerably among the batches independently of the
formulation. Hierro, de la Hoz, and Ordoñez (1997)
have found similar increases in FFA of fermented sau-
sages, with a relative decrease of saturated acids, a
relative increase of monounsaturated ones (oleic) and a
more pronounced rise of polyunsaturated fatty acids.
The lipolytic activity was ascribed primarily (about
70%) to meat tissue lipases whereas lipolytic starters,
such as Micrococci, were credited as playing mainly the
role of reducing nitrates and nitrites.
Molly et al. (1996) had, also, come to the conclusion
that lipolysis is almost exclusively brought about by
muscle and fat tissue enzymes and that Micrococcaceae
do not increase overall lipolytic activity. Poly-
unsaturated fatty acids (o-6 linoleic acid) are liberated
mainly from the polar lipid fraction, a point also shared
by Alasnier et al. (1998) and by Martı́n et al. (1998), and
their hydrolysis is higher than for monounsaturated and
saturated fatty acids. The importance of endogenous
lipase activity and the scarce effect of Micrococcaceae
on lipolysis was confirmed by Molly et al. (1997).
According to the same researchers, 60–80% of total
lipolysis is brought about by endogenous muscle
enzymes with variations deriving from individual bat-
ches and depending on the strain of Micrococcaceae
added. Lipolysis seemed to be more important in pork
compared to beef, whereas degree of comminution
420 E. Zanardi et al. / Meat Science 66 (2004) 415–423

Table 5
Free fatty acids content (mg/g fat) of M1–F1, M1–F2, and M1–F3 fresh mince and matured sausages (mean valuesstandard deviation)a

Formulations

M1 M1–F1 M1–F2 M1–F3

Mince Matured Mince Matured Mince Matured Mince Matured

C10:0 0.160.01 0.230.07 0.13 0.04 0.16 0.08 0.120.01 0.190.01 0.14 0.01 0.130.11
C12:0 0.060.01 0.190.07 0.07 0.01 0.11 0.02 0.060.02 0.120.01 0.08 0.01 0.140.01
C14:0 0.240.01 0.690.06 0.19 0.03 0.55 0.05 0.190.01 0.520.09 0.27 0.01 0.640.18
C16:0 2.810.06 6.160.82 1.66 0.02 4.74 0.49 2.040.16 4.580.11 3.34 0.60 5.961.47
C16:1 0.220.02 0.750.13 0.14 0.01 0.54 0.01 0.160.03 0.550.06 0.29 0.02 0.740.18
C18:0 1.630.01 3.210.26 1.32 0.02 2.49 0.17 1.420.09 2.500.04 2.02 0.39 3.080.61
C18:1 3.240.11 11.62.00 2.23 0.01 8.47 0.44 2.590.44 8.230.32 4.41 0.59 10.82.18
C18:2 1.350.04 6.470.53 0.94 0.02 4.80 0.16 1.060.13 4.510.12 1.70 0.12 5.571.14
C18:3 0.080.02 0.360.12 0.08 0.01 0.28 0.03 0.100.02 0.220.01 0.12 0.02 0.320.06
C20:1 0.080.03 0.480.02 n.d. 0.09 0.12 n.d. 0.280.05 n.d. 0.450.10
C20:2 0.120.01 0.790.16 n.d. 0.30 0.06 n.d. 0.340.07 0.21 0.02 0.500.04
C20:4 0.110.01 0.800.30 n.d. 0.46 0.02 n.d. 0.730.09 n.d. 0.600.06
Total FFA 10.10.14 31.73.43 6.76 0.07 23.0 1.11 7.730.33 22.80.72 12.6 1.73 28.95.84
Saturated FFA 4.890.05 10.51.15 3.37 0.03 8.05 0.51 3.820.29 7.900.24 5.85 1.00 9.942.16
Mono FFA 3.550.16 12.82.11 2.37 0.01 9.10 0.55 2.750.47 9.060.33 4.70 0.61 12.02.46
Poly FFA 1.660.04 8.400.18 1.02 0.03 5.83 0.05 1.160.16 5.800.14 2.02 0.12 6.991.22
Total FFA matured/ 3.10.4ab 3.40.2a 2.3 0.2b
total FFA mince 3.00.1ab
Sat. FFA matured/ 2.10.2a 2.40.2a 1.7 0.1a
sat. FFA mince 2.10.2a
Mono FFA matured/ 3.60.8a 3.80.3a 2.5 0.2a
mono FFA mince 3.30.5a
Poly FFA matured/ 5.10.2ab 5.70.2a 3.4 0.4b
poly FFA mince 5.00.6ab
a
Different letters, in a line, stand for significant differences, P40.05, Scheffè’s test; n.d.: not detectable.

should enhance lipolytic activity. Kenneally, Schwarz,
Fransen, and Arendt (1998) observed that there were no
differences between sausages inoculated with lipolytic
starters and those inoculated with non lipolytic ones.
Differences were observed between different trials,
probably due to differences in raw materials and varia-
tions of the latter could have more effect on lipolysis
than starter bacteria.
As for the role played by additives and technological
conditions, Motilva and Toldrà (1993) found that acid
lipase activity was activated by decreasing water activity
and by increasing salt concentrations. The latter point
was not shared by Stahnke (1995) who reported a sig-
nificant negative effect of salt on FFA. Nitrate did not
seem to affect lipase and esterase activities significantly
whereas ascorbic acid had a limited inhibitory activity,
according to Motilva et al. (1993), but nitrite, by inhi-
biting certain microrganisms, could affect their lipolytic
activity (Stahnke, 1995). Aguirrezábal, Mateo, Dom-
ı́nguez, and Zumalacárregui (2000) argued that, in
Spanish dry sausages, the increase of free fatty acids
during ripening was not dependent on the presence/
absence of spices (garlic and paprika) and/or curing
agents (nitrite, nitrate and ascorbic acid). Toldrá and
Flores (1998) also reported that curing agents did not
affect lipolysis in dry cured ham, although the same
authors observed that the presence of ascorbic acid
produced a slight inhibition on enzyme activities. Mus-
cle lipases seemed to be favoured by lower pH (Toldra,
1992), a condition found in northern type sausages,
whereas Micrococcaceae were more active at higher pH
(Leistner, 1992), typical of south of Europe sausages.
The production of FFA should be stimulated by high
temperature (Stahnke, 1995), and northern type
sausages are processed at higher temperatures.
Finally, differences in lipolytic activity could also
derive from the presence of surface moulds (in fer-
mented products of south of Europe). Bruna, Ordóñez,
Fernández, Herranz, and de la Hoz (2001) have pointed
out that surface moulds have significant lipolytic activ-
ity and play important roles against lipid oxidation.
The combined analysis of the results obtained with
the seven formulations produced in the present study,
and of the data available in the literature, confirms that
lipolysis in dry fermented meat products is mainly due
to endogenous lipases and is largely independent of
processing conditions. Indeed, differences in raw mate-
rials (pork, beef, type of fat, fresh or frozen raw mate-
rials), additives, spices, degree of comminution, size of
sausages, starter cultures, pH, processing temperatures
and length of maturing do not appear to be related to
variations observed in free fatty acids from the minces
 E. Zanardi et al. / Meat Science 66 (2004) 415–423
Table 6
Total cholesterol (mg/100 g), cholesterol oxides (COP) (mg/g) and TBARS (mg MDA/kg) content of matured sausages (mean valuesstandard
deviation)
M1 M2 N1
Cholesterol 94.5 3.5 97.01.8 66.31.8
7b-Hydroxy cholesterol 0.19 0.03 0.520.50 0.190.11
5,6a-Epoxycholesterol 0.44 0.10 0.750.22 0.610.13
7keto-Cholesterol 0.08 0.05 n.d. n.d.
Total COP 0.71 0.11b 1.270.54ab 0.790.14b
% Oxidised cholesterol 0.08 0.02b 0.130.05ab 0.120.02b
TBARS 0.10 0.01cd 0.040.01d 0.050.02d
n.d.: not detectable.
to matured products. The differences found among the
various formulations appear to be more probably due to
inherent variations of the lean and fat tissues used. It
has been reported, for instance, that slaughter condi-
tions could influence the degree of the glycerides
hydrolysis due to activation of the triglyceride lipase by
adrenalin released (Girard, Goutefongea, Monin, &
Touraille, 1986). Also, recent work has clearly shown
differences in muscle lipase activity associated with dif-
ferences in animal genetics (Claeys et al., 2001).
A further element that should be kept in mind is the
decrease during the final stages of maturation of some
free unsaturated fatty acids, such as linoleic acid,
reported by other researchers (Coutron-Gambotti, &
Gandemer, 1999; Demeyer et al., 1974; Fernández, de la
Hoz, Dı́az, Cambero, & Ordoñez, 1995). In the present
investigation free fatty acids have not been determined
at intermediate phases of processing and the four Eur-
opean types differed significantly for total maturation
time (from 14 days of N1 to 40 days of M1). It cannot
be said, therefore, if such a phenomenon has taken place
but, taking into account that processing time was very
short in all cases, compared to dry ham, the data on
lipid oxidation (see below) could suggest that something
in that line might have occurred.
3.3. Lipid oxidation
Cholesterol oxidation in matured sausages, as shown
by the three oxides observed in all samples, 7b-hydroxy-
cholesterol, 5,6a-epoxycholesterol and 7keto-choles-
terol, was low, varying from a minimum of 0.08% to a
maximum of 0.2%. Values lower than 0.1% of oxidised
cholesterol were observed in M1, N2, M1–F1 and M1–
F2 whereas M2 and N1 were slightly higher than 0.1%
and M1–F3 was on an average of 0.2% (Table 6). The
measurements of TBARS have shown that standard
formulations M1, M2, N1 and N2 did not exceed the
value of 0.10 mg MDA/kg, whereas formulation M1–F1
was slightly higher (0.15 mg MDA/kg), M1–F2 was
significantly higher (0.30 mg MDA/kg) and M1–F3 was
421
Formulations
N2 M1–F1 M1–F2 M1–F3
87.12.4 94.13.6 88.610.1 88.02.4
0.100.02 0.180.09 0.090.04 0.730.11
0.640.16 0.470.16 0.410.14 0.440.12
n.d. 0.170.10 0.140.03 0.580.07
0.750.17b 0.810.34b 0.640.14b 1.740.23a
0.090.02b 0.090.04b 0.070.02b 0.200.03a
0.070.01d 0.150.02c 0.300.04a 0.220.01b
just above 0.2 mg MDA/kg. TBARS values of all for-
mulations, though, were lower than the suggested
threshold for the appearance of rancidity off flavours in
fresh pork (0.5 mg MDA/kg) (Lanari, Schaefer, Cas-
sens, & Scheller, 1995) and in cooked meat (1.0 mg
MDA/kg) (Igene et al., 1979).
The data just reported agree with previous measure-
ments of cholesterol oxidation and TBARS in a typical
Italian dry fermented meat product (Ghiretti et al.,
1997; Novelli et al., 1998; Zanardi et al., 2000). Choles-
terol oxidation in normal conditions varied up to and
around the value of 0.1% of total cholesterol (Zanardi
et al., 2000) and the oxides constantly observed were the
three, 7b-hydroxycholesterol, 5,6a-epoxycholesterol and
7keto-cholesterol, found also in the present study.
Thiobarbituric acid reactive substances have always
been found to be lower than 0.50 mg MDA/kg in sau-
sages produced with fresh raw materials. Therefore,
values up to 0.10 mg MDA/kg, such as those observed
in M1, M2, N1 and N2, can be considered equivalent,
irrespective of possible statistical differences, taking into
account an acceptable level of variation due to raw
materials and processing conditions.
A concentration of oxysterols around 0.1% is approxi-
mately the minimum level for toxicity to be observed in
‘‘in vitro’’ experiments on cultured cells but about 100
times lower than the level required to show toxic effects in
‘‘in vivo’’ trials with laboratory animals (Bösinger, Luf, &
Brandl, 1993). The value of 0.2% found in the formula-
tion (M1–F3) devoid of antioxidants (nitrates, nitrites,
ascorbic acid) is significantly higher than the remaining six
formulations but it is probably more significant for its
proximity to normal levels. The ‘‘normal’’ value of for-
mulation M1–F2 is also notable. Such results appear to
suggest that in fermented meat products, in spite of the
mincing and mixing of different raw materials, ingredients
and additives, oxidation is remarkably curbed by reducing
systems of the animal tissues and, perhaps, by bacterial
growth with the consumption of oxygen that it entails.
Antioxidant properties of garlic and paprika in dry
fermented sausages have been reported (Aguirrezábal et
422 E. Zanardi et al. / Meat Science 66 (2004) 415–423
al., 2000) but were probably due to the high amounts
used (3% paprika, 1% garlic). Garlic, white and black
pepper in the present investigation were employed at
total doses lower or not much higher than 0.1%, suffi-
cient for flavour purposes but it is doubtful that they
could develop an antioxidant activity comparable to
nitrite. The antioxidant effect of nitrite is probably due
to a chelating action of nitrite towards non-haem iron
released during chopping and mincing of raw materials
(Morrissey et al., 1985) whereas spices should act as free
radicals scavengers in a similar manner as ascorbic acid.
4. Conclusion
The different formulations used to produce fermented
sausages representative of the main types existing in
Europe have provided data which support the hypoth-
esis that most of the proteolysis and lipolysis taking
place during maturation is of endogenous origin. Tech-
nology appears to affect proteolysis indirectly: differ-
ences in rate of pH decline and final pH may affect
endogenous proteolytic enzymes. Other technological
parameters, such as starter cultures, additives and spices
do not seem to have perceivable effects on lipolysis and
lipid oxidation.
No significant differences have been observed in lipid
oxidation among different formulations with the only
exception of the formulation lacking nitrites/nitrates
and ascorbic acid. Such a result stresses the need for
some antioxidant compounds in the formulation not
only for colour stability purposes but also for lipid
protection and, therefore, for the safety of matured
products.
Acknowledgements
Research financed by the European Commission
(Contract FAIR CT97-3227 ‘‘Bioflavour’’).
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