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Meat Science 79 (2008) 784–794
MEATSCIENCE
Proteolysis in Painho de Portalegre dry fermented sausagein relation to ripening time and salt content
L.C. Roseiro *, C. Santos, M. Sol, M.J. Borges, M. Anjos, H. Gonc�alves, A.S. Carvalho
Instituto Nacional de Engenharia, Tecnologia e Inovac�ao, DTIA, Estrada do Pac�o do Lumiar, 1649-038 Lisboa, Portugal
Received 22 February 2007; received in revised form 8 November 2007; accepted 18 November 2007
Abstract
The effect of salt addition (3% and 6% in the final product) on the shelf-life related physicochemical characteristics and proteolysisprofile during the ripening period of a Portuguese dry fermented sausage ‘‘Painho de Portalegre”, were evaluated. The product with6% salt concentration had low aw and pH values at most ripening periods evaluated, due to the influence of NaCl on the water bindingcapacity of the protein structure and to the low ammonia accumulation, respectively. Similar changes were observed for total basic vol-atile nitrogen (TBVN), free amino acid nitrogen (FAAN) and non-protein nitrogen (NPN) fractions in both products. After a clearincrease during the first days of the processing phase, all the initial rates slowed down with some fluctuation in FAAN and NPN. Inrelation to small peptides and free amino acid accumulation, the major differences between the tested formulations were mainly observedon distinct profiles rather than on overall concentrations.� 2007 Elsevier Ltd. All rights reserved.
Keywords: Dry fermented sausage; Ripening; Salt addition; Proteolysis; Peptides; Free amino acids
1. Introduction
Distinct cultural and social backgrounds of the popula-tions and the environmental/climatic conditions in the Por-tuguese geographical regions, determine greatly thephysical and sensorial characteristics of each country styledry-fermented meat product. A great effort has been madeto promote specific processed meat products (ProtectedGeographical Indication – PGI and Protected Designationof Origin – PDO) amongst the traditional production.
The main processing mechanism for preserving purposesin artisanal dry fermented sausage production (no additivesand starter addition) is based on aw reduction of the prod-uct to about 0.87. This aw reduction is achieved throughboth dehydration promoted during drying/smoking andsalt addition to the formulation. Variable NaCl levels, upto 7% in the final product (wet basis) have been detected
0309-1740/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2007.11.012
* Corresponding author. Tel.: +351 217127107; fax: +351 217127162.E-mail address: [email protected] (L.C. Roseiro).
in some products. Proteolysis occurring during the differentprocessing and storage stages is extremely important forthe development of the final texture and taste/flavour attri-butes, due to the formation of small molecular compo-nents, mainly polypeptides, smaller peptides, amino acidsand amines, known as taste promoters and flavour precur-sors (Demeyer et al., 1995; Ordonez, Hierro, Bruna, &Hoz, 1999; Stahnke, 2002, 2003). These reactions, whichare mainly the result of endogenous enzymes (Pezacki &Pezacka, 1986; Toldra, Rico, & Flores, 1992), are affectedby several processing factors, including the salt content ofthe formulation. Due to the connection with high bloodpressure and coronary heart diseases, the reduction ofsodium concentrations in traditional meat products wouldbe very desirable. In case of uptake of such a technologicaloption, through addition of less NaCl, the pattern of pro-teolysis can be expected to change since it will influenceendogenous enzymes and microbial activity.
The present study reports the proteolysis results of twodifferent ‘‘Painho de Portalegre” batches prepared with
L.C. Roseiro et al. / Meat Science 79 (2008) 784–794 785
two different salt concentrations. The samples were testedat different times during ripening up to 180 days, afterthe filling operation.
2. Materials and methods
2.1. Sausage preparation
‘‘Painho de Portalegre” dry fermented sausages weremanufactured in the plant of a local small factory, usinglean pork and fat from the autochthonous ‘‘Alentejano”
pig breed, reared on an extensive production system. Thecomposition of the formulation (%, w/w) was lean porkham and shoulder (80); belly or back fat (20). The otheringredients were calculated in relation to raw materialweight, amounting as follows: salt (3% or 6%); paprikapaste (5.8%); raw garlic paste (0.56%) and tap water(14%). After grinding the meat and the fat to a size ofabout 2 cm (with adjustable plate holder diameter set),raw materials were mixed for about 5 min, during whichNaCl, paprika paste, raw garlic paste and tap water wereadded. No nitrate/nitrite, sugar and starter culture wereused. This seasoned batter was kept in a refrigerator (0-2 �C) for three days, to promote the interaction betweeningredients. After this period, the batter was stuffed intonatural pig casings (approximately 25 cm long and 5 cmin diameter), using a piston filler. Visible air bubles betweenthe paste and the casing were removed by manual punctur-ing. To remove the excess of water from casings, the rawsausages stood for about 1 h at room temperature (10–12 �C). Only then they were placed in a traditional smok-ing/drying room.
Two specific batches were prepared in two distinct trials,processed as described before, but with different NaClamounts added, in order to reach, approximately, 3.0%and 6.0% in the final product (wet basis). During the smok-ing/drying phase product weight losses reached 40%. Thisvalue was used to calculate the salt added to the formula-tion. Sausage preservation was ensured by some acidificat-ion achieved through naturally occurring lactic acidbacteria activity, but mostly by aw reduction to around0.87.
2.2. Samples
Sampling included raw meat/fat mixtures immediatelyafter grinding and mixing (R0), seasoned raw materialskept for 3 days in the refrigerator at 0–2 �C (R1), andstuffed product with 6 (S1), 15 (S2), 40 (S3) days of pro-cessing in a traditional drying and smoking house. Undergood weather conditions, the last sample (S3) correspondsto the final processed product. To reproduce the effect of alonger ripening period, S3 samples were held at room tem-perature (15 �C ± 2 �C) under vacuum packaging duringfurther storage periods of 50 (S4), 80 (S5) and 140 (S6)days.
2.3. Physicochemical analysis
Water activity (aw) of samples was measured in aRotronic Hygromer at 20 �C with a probe AwV C-DIO(Rotronic AG, Switzerland). The pH was determined witha 654 pH meter (Metrohm Herisau, Switzerland) equippedwith combined pH glass electrode (Mettler Toledo, Swit-zerland). Dry matter (DM) was determined by drying thesample at 103 �C ± 2 �C to constant weight.
To analyse nitrogen fractions, 25 g of sausage werehomogenized with 90 mL distilled water and 25 mL of25% (w/v) trichloroacetic acid solution in a Polytronhomogenizer Model PT 3100 (Kinematica AG, Switzer-land). The samples were left at +4 �C for 2 h, filtered andmade up to 200 mL with distilled water. Non-protein nitro-gen (NPN) was determined from 50 mL of filtrate by theKjeldahl method in a Kjeldahl Foss automatic system.The free amino acids nitrogen (FAAN) determination used25 mL of filtrate neutralized with 1.5 N NaOH solution.Then 10 mL of 40% (v/v) formaldehyde solution wereadded and neutralized, once more, with 0.02 N NaOHsolution. Total volatile basic nitrogen (TVBN) determina-tion was carried out by the Conway microdiffusion tech-nique (Pearson, 1973). The nitrogen fractions content wasexpressed as g/100 g dry matter of sample.
2.4. Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE)
Myofibrillar proteins were extracted by the methoddescribed by Toldra, Rico, and Flores, 1993 and then ana-lysed by polyacrylamide gel electrophoresis, using poly-acrylamide gel gradient electrophoresis (SDS-PAGGE)using Multiphor II Electrophoresis Unit and ExcelgelSDS Gradient 8–18 (Amersham Pharmacia Biotech)according to the instructions supplied by the manufacturer.SDS-PAGE was performed on 20% homogeneous gelsaccording to the instructions enclosed. Gels were stainedwith Coomasie Brilliant Blue G-250 and scanned with anImageScanner (Umax PowerLook III), LabScan v3.00and ImageMaster 1D Elite Software v3.0. Scans were ana-lysed quantitatively using Image Master 1D Elite 4.0(Amersham Pharmacia Biotech). The molecular weight ofproducts from proteolysis was estimated by reference tothe relative mobilities of standard proteins (Low and HighMolecular Weight Calibration Kits for SDS-PAGE, Amer-sham Pharmacia, Biotech): myosin (220 kDa), a2-macro-globulin (170 kDa), b-galactosidase (116 kDa), transferrin(76 kDa), glutamic dehydrogenase (53 kDa), phosphorilase(97 kDa), albumin bovine serum (66 kDa), ovalbumin(45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor(20.1 kDa), a-lactalbumin (14.4 kDa).
2.5. Peptides
Extraction of 20 g of sample was performed 3 times with45 mL of trichloromethane:methanol (2:1) in an orbital
Tab
le1
Ch
ange
sin
aW
,p
H,
nit
roge
nfr
acti
on
s(e
xpre
ssed
asg/
100
go
fD
M)
and
amm
on
ia(e
xpre
ssed
asm
g/10
0g
of
DM
)d
uri
ng
the
rip
enin
go
f‘‘
Pa
inh
od
eP
ort
ale
gre
”m
anu
fact
ure
dw
ith
3%an
d6%
of
NaC
lin
fin
alp
rod
uct R
ipen
ing
stag
esS
EE
ffec
ts
R0
R1
S1
S2
S3
S4
S5
S6
3%6%
3%6%
3%6%
3%6%
3%6%
3%6%
3%6%
RT
SR
Tx
S
pH
6.18
5.84
a5.
87a
5.50
ef5.
71b
5.31
h5.
13i
5.49
ef5.
36gh
5.59
cd5.
37gh
5.61
c5.
41g
5.54
de
5.47
f0.
01**
***
***
*a
Wn
d0.
95a
0.95
ab
0.94
ab
0.93
bc
0.92
c0.
92c
0.87
d0.
83e
0.86
d0.
80f
0.87
d0.
83e
0.83
e0.
86d
0.00
3**
***
***
*A
mm
on
ia43
.751
.974
.215
1.0
105.
818
3.9
153.
921
1.3
184.
522
9.6
199.
026
2.2
212.
324
8.1
222.
117
.4**
***
ns
NP
N0.
590.
61i
0.96
a0.
75f
0.74
fg0.
58j
0.72
gh
0.90
c0.
94b
0.78
e0.
47k
0.90
c0.
94b
0.85
d0.
71h
0.00
4**
***
***
*F
AA
N0.
080.
09i
0.07
j0.
19h
0.17
h0.
29e
0.22
g0.
36b
0.32
cd0.
33c
0.27
f0.
33c
0.36
b0.
38a
0.31
de
0.00
3**
***
***
*T
VB
N0.
030.
03h
0.02
h0.
05fg
0.05
g0.
08d
e0.
07e
0.11
bc
0.10
cd0.
07ef
0.07
ef0.
12b
0.11
c0.
13ab
0.14
a0.
003
***
****
*
‘‘In
sam
ero
w,
mea
ns
wit
hd
iffer
ent
lett
ers
are
sign
ifica
ntl
yd
iffer
ent”
.R
T–
rip
enin
gti
me
effec
t;S
–sa
lteff
ect.
ns
=n
ot
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isti
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ysi
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=p
<0.
01,
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001.
nd
–n
ot
det
erm
ined
.
786 L.C. Roseiro et al. / Meat Science 79 (2008) 784–794
shaker at 400 rpm for 10 min. Then, after vacuum filtrationthrough Whatman no. 4 filter paper, the extracts weremixed with water in a separating funnel, and left overnight.The aqueous phase was evaporated at 40 �C under vacuumand re-suspended with distilled water. The water solubleextract had a concentration of approximately 0.2 g/mL ofpeptide and was kept at �80 �C until gel separation.
Peptide extracts were previously filtered through a0.22 lm membrane before being injected (100 lL) on theAKTA FPLC system, equipped with an UV detector(214 nm). The gel separation was performed on a superdexpeptide HR 10/30 column (Amersham Pharmacia Biotech),10 � 300–310 mm, with a total bed volume of approxi-mately 24 mL and a pH stability of 1–14, using as eluent0.25 M NaCl in 0.02 M phosphate buffer, pH 7.2, filteredin advance by 0.22 lm membrane. The separation was per-formed at a flow rate of 1.0 mL/min in about 26 min.
2.6. Free amino acids
Free amino acids and ammonia were determinedaccording to the Directive 98/64/EC, using an amino acidanalyzer Biochrom 20 (Pharmacia Biotech). The freeamino acids were extracted with diluted hydrochloric acid.Coextracted nitrogenous macromolecules were precipitatedwith sulfosalicylic acid and then removed by filtration.After this procedure, pH was adjusted to 2.2 and the aminoacids separated by ion exchange chromatography. Freeamino acid contents were determined by reaction with nin-hydrin with photometric detection at two wavelengths,570 nm and 440 nm (for proline) and expressed as mg/100 g DM of sample. In the text, the terms ‘‘sweet”, ‘‘bit-ter”, ‘‘acidic” and ‘‘aged” flavour amino acids correspondto the sum of glycine, alanine, serine, threonine, praline,hydroxyproline; histidine, arginine, methionine, valine, leu-cine, isoleucine, phenylalanine; glutamic acid, aspartic acid,histidine, asparagine and lysine, tyrosine, aspartic acid,respectively.
2.7. Statistical analysis
The influence of ripening period and salt concentrationon data was evaluated by two-way ANOVA (JMP, TheStatistical Analysis Software – SAS Institute, 2002). TheTukey HSD post hoc test was used for comparison ofmeans values. Differences were considered significant atp < 0.05.
3. Results and discussion
3.1. Physicochemical parameters
The pH values from raw material mixtures decreasedquickly during the fermentation phase (up to day 15 ofthe processing stage), falling from about 6.1 to 5.3 and5.1 in 3% and 6% formulations, respectively (Table 1). Inboth products the pH slightly increased thereafter (<0.5
L.C. Roseiro et al. / Meat Science 79 (2008) 784–794 787
units), mostly until the 90th day of ripening, and then sta-bilized around 5.5. When statistically significant (p < 0.05),pH differences between formulations presented lower val-ues in 6% ‘‘Painho de Portalegre”. Despite the lower overallorganic acids production detected in these samples, whichis most evident as lesser amounts of acetic, butyric andpyruvic acids (data not shown), such pH evolution couldbe related to a possible impact of 6% salt concentrationon the natural flora (Roseiro, Santos, Sol, Silva, & Fernan-des, 2006), selecting and promoting the development ofstrains having lower ammonia production ability (Selgas,Garcia, Garcia de Fernando, & Ordonez, 1993) (Table 1).In fact, the ammonia formation rate was intense duringthe fermentation stage in both products, with the concen-tration increasing slightly but steadily up to the end ofthe tested ripening period. However, the lower salt producthad higher mean ammonia concentrations (p < 0.05).These differences ranged between a maximum of +42% atday 6 and a minimum of +14.5% at day 40. Regardlessof the salt content, ‘‘Painho de Portalegre” sausage is alow acid fermented product, with its pH profile determin-ing the speed of the drying process and, possibly, promot-ing a lower hydrolytic action by cathepsin D anddipeptidylpeptidases I and II (Toldra, 2006a), which willinfluence taste, flavour (Henriksen & Stahnke, 1997) andcolour formation as well. Diaz, Fernandez, Garcia de Fer-nando, Hoz, and Ordonez (1993, 1996) observed similarpH profiles in other Iberian dried fermented traditionalsausages.
The changes in aw and weight loss along the ripeningperiod showed the usual trends observed in this type ofproducts (Fig. 1). Despite the similarity of moisture lossdynamics in both products, the 6% salt formulationshowed, comparatively, lower aw values (p < 0.05) at theend of processing and in the early storage stages (Table1). This trend could be determined by the influence of NaClon the muscle proteins structure, traduced as higher waterbinding capacity. Most muscle enzymes involved in prote-olysis of processed dry meat products are strongly affectedby the decrease in aw, which has a particular impact in
Fig. 1. Changes in weight losses (WL) and aw during the ripening of‘‘Painho de Portalegre” manufactured with 3% and 6% of NaCl in finalproduct.
cathepsins and aminopeptidases (alanyl and pyroglutamyl)performance (Toldra, 2006b). In addition, the level of saltalso partially inhibits muscle and microbial tri/dipeptidyl-peptidases and aminopeptidase activities (Sentandreu &Toldra, 2001; Toldra et al., 1992; Toldra et al., 1993). Inthis sense, 6% ‘‘Painho de Portalegre” should have lowerconcentrations of small peptides and free amino acidsand consequently, to have lower nitrogen fractions levelsthan the 3% counterpart.
Excluding the effect of salt concentration for TVBN(p < 0.01), the influence of each factor or their interactionswas highly significant (p < 0.001) for the other nitrogenfractions (Table 1). The evolution patterns of FAAN andTVBN along the 180 days ripening period, were similar,both increasing about 4 times over the initial concentrationpresent in raw material mixtures (0.08 g/100 g DM and0.03 g/100 g DM vs 0.33 g/100 g DM and 0.135 g/100 gDM, respectively). This is in agreement with other reportsof the proteolytic activity of dry cured meat products(Motilva, Toldra, Nieto, & Flores, 1993; Toldra, 1998;Toldra, Aristoy, & Flores, 2000; Toldra, 2006a). Regardingthe NPN fraction, during processing and storage periodssome variation was observed in concentrations. This couldbe due to the characteristic multi-step development of theproteolysis chain, from proteins to polypeptides and smallpeptides down to free amino acids (Toldra et al., 2000).Concerning the effect of decreasing salt level on the prote-olysis intensity referred by several authors, this trend wasmostly confirmed in relation to FAAN. In relation to theTVBN fraction this effect was almost not found since differ-ences were not significant (p > 0.05) and diverged for mostripening stages in NPN. The poor control of the tempera-ture homogeneity in the smoking/drying room, frequentlyobserved in traditional production, could also be thesource of some counterbalance to the NaCl inhibition effect(Martin, Cordoba, Antequera, Timon, & Ventanas, 1998;Toldra et al., 1993; Toldra, Cervero, & Part, 1993; Virgili,Parolari, Schivazappa, Soresi Bordini, & Borri, 1995), con-tributing to the atypical evolution of the NPN fraction inthe present study. Nitrogen fractions concentration duringthe ripening time did not differ from those reported in otherstudies (Diaz et al., 1996; Cantoni, D’Aubert, & Bresclani,1985).
3.2. SDS-PAGE
During the fermentation and ripening of dry fermentedsausages a large number of biochemical reactions associ-ated to the degradation of myofibrillar and sarcoplasmicproteins take place (Diaz et al., 1996). These biochemicalreactions are promoted by muscle endopeptidases (calpainsI and II and cathepsins B, D, H and L) (Toldra, 2006b) andmicrobial proteinases, bound either to the cell wall or tothe cell membrane (Visser, 1993). The electrophoretic pro-files of myofibrillar proteins of ‘‘Painho de Portalegre”, atdifferent processing and storage stages, for formulationswith 3% and 6% salt in the final product, are shown in
Fig. 2. SDS-PAGE electrophoretograms of myofibrillar protein extracts throughout the ripening of ‘‘Painho de Portalegre” manufactured with 3% (a) and6% (b) of NaCl in final product. Lane Std, molecular weight standards ranging from 14 to 220 kDa.
788 L.C. Roseiro et al. / Meat Science 79 (2008) 784–794
Fig. 2. Regarding myosin (220 kDa), an important degra-dation process took place in both salt levels, with most ofthis hydrolysis occurring up to the 6th day of drying/smok-ing, indicating a more intense development in the less saltedformulation (�82% vs �70%). The extension of myosinhydrolysis also differed between the two products. Less saltresulted in a continuous decrease in staining intensity val-ues during the entire duration of ripening (120 days), incontrast with that of 6% salt addition, which remainedalmost unchanged after the 40th day of the drying/smokingoperation. Excepting the results obtained with R1 samples,the degradation of myosin presented an inverse relation-ship with the salt level in the product. This trend confirmedthe results obtained in other studies (Aristoy & Toldra,1991; Toldra, 1992) and demonstrates the inhibition ofcathepsins by salt (Toldra, 1998; Toldra et al., 1992).
The hydrolysis of actin (45 kDa) was less intensive thanthat of myosin, with the maximum extraction obtainedfrom the 3% salt ‘‘Painho de Portalegre” samples ripenedfor 120 days (about �39%). The hydrolysis dynamic alsodiffered between the two formulations. In 3% salt ‘‘Painho
de Portalegre” samples the hydrolysis started at a very slowrate at day 15 of drying/smoking (�5.6%), increasing thenup to day 40 (about �22.1%), remaining almost unchangeduntil day 90 and increasing again at day 120 (�39%). Incontrast, 6% ‘‘Painho de Portalegre” hydrolysis patterndid not show any relevant change up to day 40, then fellto approximately �37% until day 90.
A wide variety of polypeptides and large peptides werealready extracted from R0 samples, ranging in molecularweight from 16.5 kDa to almost 188 kDa (Table 2).Throughout the ripening period of both products 12 newcomponents were detected, with high (207 and 142 kDa),medium (75, 61, 54.780, 47.673, 44 kDa) and low (33,29.4, 15.368, 13.808 and 11.456 kDa) molecular weights,most of them appearing soon after the seasoning operation(R1) or during the fermentation stage in the smoking/dry-ing room (S1). This overcomes what Johansson, Berdague,
Larson, Trane, and Borch (1994) reported, probablyreflecting different enzyme potential of raw materials anddistinct processing conditions applied in the manufactureof the products studied (Armero, Baselga, Aristoy, & Told-ra, 1999a; Armero, Barbosa, Toldra, Baselga, & Pla,1999b; Rosell & Toldra, 1998; Toldra, Flores, Aristoy, Vir-gili, & Parolari, 1996). Compounds with lower molecularmass, which are believed to make important contributionsas flavour compounds or precursors (Cordoba et al., 1994),showed, when comparing gel electrophoresis, relevantincreases on their band density, at different stages of theprocess (Table 2). The sum of components with molecularweights up to 41 kDa, extracted after 6 days of drying/smoking, represented close to 78% and 70% of their overallevolution (S5-120 days), for 3% and 6% ‘‘Painho de Portal-
egre”, respectively. This faster formation during the fer-mentation stage might be related to the influence that theincrease in environmental temperature has on the endoge-nous enzymes activity, mainly cathepsin D (Molly et al.,1997; Toldra, 2006a; Verpaletse, Demeyer, Gerard, &Buys, 1992), although proteolysis by bacteria and yeastspresent in the batter should not be ignored (Bolumar,Nieto, & Flores, 2001; Martin et al., 1998; Rodriguez,Nunez, Cordoba, Bernudez, & Asensio, 1998). The overallproteolysis taking place in the ‘‘Painho de Portalegre” sau-sages with 3% salt is comparatively more pronounced, withthe difference depending on the processing stage or thelength of the storage period.
3.3. Peptides
Peptides resulting from protein breakdown have beenassociated with specific tastes in dry cured meat products(Aristoy & Toldra, 1995). The different eluted small pep-tides (<7000 Da) from ‘‘Painho de Portalegre” sausages,comprising six different peaks, are shown in Table 3. Theinfluence of ripening time was significant for all detectedpeptide groups (p < 0.001 for peaks 1, 2, 3, 5 and 6;
Table 2Relative density (%) of the electrophoretic bands obtained for myofibrillar proteins extracted from ‘‘Painho de Portalegre” manufactured with 3% and 6%of NaCl, along the ripening period
MW (kDa) R0 3% 6%
R1 S1 S2 S3 S4 S5 R1 S1 S2 S3 S4 S5
220.000 46.24 22.07 8.32 6.17 3.50 4.30 2.17 21.56 13.93 9.30 6.87 8.34 7.80206.918 1.18 1.21 0.88 1.12 0.59 1.94 1.36 0.82 0.95 0.79188.002 0.96 1.31 1.70 1.65 1.44 1.64 1.44 1.35 1.87 2.07 1.85 1.74 1.67168.598 4.11 2.46 2.87 3.32 3.06 3.55 3.50 2.70 2.85 3.14 3.82 4.05 3.38155.800 2.42 5.44 8.38 5.49 6.53141.975 4.19 4.88 12.54 15.49 14.09 16.70 3.91 4.19 8.60 8.78 11.91 9.84124.979 1.89 1.32 0.82 1.10 0.67 1.20 1.16 1.63 1.24 1.08 0.79 1.00 0.95115.134 1.05 0.95 0.75 0.99 0.82 0.96 0.72 1.03 1.01 0.91 0.57 0.78 0.94109.961 0.52 0.69 0.40 0.47 0.51 0.55 0.72 0.54 0.57 0.56 1.00101.450 2.21 2.35 2.84 2.98 3.20 2.92 2.83 2.78 2.56 3.29 3.69 2.89 2.9297.000 0.42 1.56 1.66 2.11 2.61 2.60 2.79 2.31 1.72 3.15 3.49 3.77 4.0286.310 0.51 0.50 0.53 0.38 0.42 0.50 0.64 0.85 0.64 0.74 0.46 0.40 0.4880.004 0.39 1.12 0.60 0.51 0.40 0.74 0.66 0.62 0.43 0.58 0.92 1.08 1.4075.040 0.30 0.44 0.44 0.57 0.48 0.26 0.38 0.50 0.54 0.74 0.7371.415 1.92 2.09 1.77 1.74 1.99 1.97 2.06 1.65 1.69 1.56 1.89 1.82 1.5569.891 0.46 0.88 0.73 0.58 0.80 0.68 0.97 1.34 0.65 0.78 0.81 1.18 1.0666.000 2.01 2.96 2.64 2.60 3.08 2.92 3.51 3.59 2.95 3.22 3.10 3.34 3.4460.715 0.72 0.91 0.86 0.78 0.79 0.41 0.70 0.80 0.78 1.8254.781 0.66 0.47 0.53 0.45 0.60 0.35 0.37 0.63 0.37 0.53 0.8552.104 1.11 1.98 1.24 1.69 1.79 1.57 1.66 1.26 1.21 1.42 1.72 1.83 1.8847.673 0.99 1.10 1.15 1.48 1.16 1.99 1.65 1.22 1.29 1.3745.000 20.64 20.18 21.01 19.49 16.08 17.06 12.55 19.98 22.78 23.82 21.88 12.84 14.9144.000 2.58 3.12 3.26 3.26 2.19 3.90 3.63 3.4142.905 0.4841.430 0.48 4.48 3.39 3.77 4.33 4.30 4.16 4.60 2.90 3.49 4.18 5.04 5.1040.001 0.60 2.70 1.33 1.83 1.70 1.83 2.25 2.07 0.91 1.32 1.13 1.08 1.0739.629 0.3637.898 0.45 1.60 2.06 2.40 2.87 2.34 2.38 1.70 1.66 1.88 2.39 2.07 2.8333.071 0.68 2.77 2.45 2.55 2.14 2.30 1.28 2.31 2.16 2.15 2.83 2.3230.639 0.76 0.69 1.29 2.14 1.49 2.33 1.88 0.77 1.94 2.42 2.36 4.2329.444 0.80 2.07 1.94 2.68 1.60 1.56 2.09 1.76 2.05 2.38 2.56 1.5427.337 0.79 0.89 1.3326.108 2.07 4.28 7.00 5.46 4.18 3.74 4.36 2.70 5.55 4.89 4.21 3.87 3.4824.646 0.56 0.75 1.45 1.73 1.89 1.99 2.89 0.96 0.77 0.84 1.20 1.38 1.6523.115 0.80 0.76 0.92 0.92 1.04 1.01 1.33 0.69 0.77 0.80 0.91 1.10 1.0021.293 1.36 2.25 2.69 2.13 2.27 2.13 2.12 2.10 2.36 2.02 2.03 1.87 1.7720.100 2.19 2.68 2.30 2.01 1.89 1.66 1.36 1.97 1.80 1.47 1.65 1.09 1.2318.793 0.58 2.42 1.82 1.54 1.28 1.53 1.21 1.90 1.50 1.54 1.41 1.26 0.9416.468 1.01 1.02 1.29 1.26 1.22 1.42 1.39 1.38 0.71 0.51 0.64 1.13 0.9715.368 0.72 0.94 1.14 0.92 1.70 0.44 0.47 0.56 0.76 0.8613.808 1.72 1.61 1.98 3.02 3.29 4.02 1.92 2.34 2.03 1.77 2.58 3.6211.456 2.18 2.48 3.50 3.60 5.16 1.27 1.28 1.97 3.55 1.98
L.C. Roseiro et al. / Meat Science 79 (2008) 784–794 789
p < 0.01 for peak 4). Salt concentration effects were, com-paratively, less expressed in relation to peak 3 (p < 0.05)and not significant in relation to peak 4. The impact ofthe interaction of factors appeared less significant in peak4 (p < 0.05), comparatively to the remaining components(p < 0.001).
Regardless of the salt content, peptides concentration inraw material mixtures represented by peak 1(597.12 mAU min) and peak 3 (69.3 mAU min) decreasedalong the processing phase, more intensively during the fer-mentation stage (S1 and S2), not in full agreement withfindings reported by Garcia de Fernando and Fox (1991).In contrast, peaks 2, 5 and 6 increased their areas, mostlyin the second half of processing, corroborating the results
obtained by Hughes et al. (2002). Also in accordance withthese authors, no new peptides were produced as ripeningprogressed. Peak 4 showed a somewhat irregular responseto factors under study. Investigations over the last decadehave confirmed cathepsin D as the enzyme primarilyresponsible for proteolysis during fermentation (Demeyeret al., 1995; Molly et al., 1997).
At the end of processing phase (S3), the effect of saltconcentration diverged in the decreasing rate obtainedfor peak 1 (�64.4% vs �69.5% in 3% and 6% salt formu-lations, repectively) and peak 3 (�95.4% vs �78.5% in3% and 6% salt formulations, repectively). Concerningthe trends observed for peaks 2, 5 and 6, 6% ‘‘Painho
de Portalegre” showed higher concentrations in the ear-
Tab
le3
Are
a(m
AU
.min
.)re
cove
ryo
fp
eak
sco
rres
po
nd
ing
toth
ep
epti
des
in‘‘
Paın
ho
de
Port
ale
gre
”m
anu
fact
ure
dw
ith
3an
d6%
of
NaC
lin
fin
alp
rod
uct
,at
diff
eren
tp
roce
ssin
gan
dst
ora
gep
erio
ds
Rip
enin
gst
ages
SE
Eff
ects
R0
R1
S1
S2
S3
S4
S5
3%6%
3%6%
3%6%
3%6%
3%6%
3%6%
RT
SR
Tx
S
Pea
k1
597.
134
6.0a
207.
5c27
0.4b
151.
2e21
9.5c
174.
9d21
2.5c
182.
3d17
8.5d
182.
0d17
8.7d
155.
8e2.
6**
***
***
*P
eak
268
.136
.7f
25.2
g60
.5e
29.7
g76
.1d
57.2
e12
2.7a
205.
3c11
7.0a
b11
5.8b
118.
6ab
112.
9b1.
1**
***
***
*P
eak
369
.376
.5a
50.8
b2.
0fg10
.6d
0.0g
4.6e
3.2ef
14.9
c0.
0g4.
6e0.
0g0.
0g0.
4**
**
***
Pea
k4
2.7
1.4c
4.8a
bc
2.5a
bc
1.6b
c2.
6ab
c1.
7bc
6.3a
5.9a
3.7a
bc
3.6a
bc
5.7a
b2.
9ab
c0.
7**
ns
*P
eak
545
.329
.1g
17.6
h64
.2ab
36.8
f54
.4d
e50
.9e
70.9
a52
.9d
e59
.6b
cd63
.7ab
c56
.3cd
e50
.5e
1.3
***
***
***
Pea
k6
4.8
3.8f
4.9f
26.6
d8.
3ef27
.8d
14.3
e37
.3b
c40
.6ab
46.4
a29
.9cd
43.6
ab
30.6
cd1.
6**
***
***
*
‘‘In
sam
ero
w,
mea
ns
wit
hd
iffer
ent
lett
ers
are
sign
ifica
ntl
yd
iffer
ent”
.R
T–
rip
enin
gti
me
effec
t;S
–sa
lteff
ect.
ns
=n
ot
stat
isti
call
ysi
gnifi
can
t,*
=P
<0.
05,
**P
<0.
01,
***
=P
<0.
001.
790 L.C. Roseiro et al. / Meat Science 79 (2008) 784–794
lier (p < 0.05) and later (p > 0.05) components whereas3% ‘‘Painho de Portalegre” samples had peak 5 with big-ger areas (p > 0.05). Throughout the storage period,peptide areas presented only small changes, indicatinga stable balance between the formation and degradationrates, with the differences between formulations beingreduced. Apparently, extending the ripening period upto 120 days over the processing phase did not seem toenhance the taste development of the product associatedwith accumulation of these peptides. Ruiz et al. (1999)reported a similar occurrence in Iberian hams, whenthe ripening process was prolonged from 420 up to600 days. Considering the overall peptide peaks eluted,6% ‘‘Painho de Portalegre” had a slightly higher concen-tration than 3% ‘‘Painho de Portalegre” in the end ofprocessing (3%–452.9 mAU min; 6%–501.9 mAU min).Those relative positions inverted after 50 days of ripen-ing under storage conditions (3%–405.2 mAU min; 6%–399.6 mAU min) and the differences strengthened whenthat period was prolonged for 80 days (3%–402.9 mAU -min; 6%–352.7 mAU min.). Since most of these proteo-lytic changes took place during the fermentation stage,microbial tri and dipeptidylpeptidases might also havea relevant role on its development.
3.4. Free amino acids
The last proteolytic step is that in which peptides areconverted to free amino acids through muscle and micro-bial aminopeptidases removing single residues sequen-tially from the N-terminus (Toldra, 1992). This actionhas utmost relevance to the overall acceptance of driedfermented meat products, since those compounds arethe source of specific tastes and odours, determining thefinal flavour characteristics (Flores, Aristoy, Spanier, &Toldra, 1997; Henriksen & Stahnke, 1997). Informationabout the variation of free amino acids will help to betterunderstand the balance between those generated andhydrolysed into volatiles and to guide producers for theuse of innovative processes, enhancing specific flavourpatterns. The profile and concentration of free aminoacids (FAA) in dry matter (DM) obtained during the pro-cessing phase and storage periods of ‘‘Painho de Portal-
egre”, manufactured with 3% and 6% salt in the finalproduct, are shown in Table 4. The effect of ripening time,salt content and the interaction of both factors on theconcentration detected for the majority of single aminoacids was significant (p < 0.001), excepting for taurine,alanine, lysine and arginine.
Total FAA increased throughout the ripening time(p < 0.001), with higher rates in the period of greatermicrobial development (up to day 15 of drying/smokingstage – S2), but was not influenced by salt content orRTxS interaction (p > 0.05). Muscle aminopeptidases,except for leucyl groups, may contribute to free aminoacids formation at this early stage since pH is not yet solow and temperature may occasionally reach 30 �C.
Table 4
Changes in free amino acid content (mg/100 g dry matter) at different processing stages and storage periods of ‘‘Painho de Portalegre” manufactured with 3% and 6% of NaCl in final product
Ripening stages SE Effects
R0 R1 S1 S2 S3 S4 S5 S6
3% 6% 3% 6% 3% 6% 3% 6% 3% 6% 3% 6% 3% 6% RT S RT x S
Tau 85.13 93.17 103.64 88.33 102.11 90.73 102.71 90.26 107.92 92.58 106.99 102.43 104.00 89.80 102.79 4.61 ns *** ns
Asp 7.33 13.91fg 10.06g 12.48g 14.06fg 43.22b 19.26ef 31.74c 23.70de 46.97b 28.23cd 44.12b 33.89c 59.88a 46.47b 1.03 *** *** ***
Thr 17.21 45.63gh 21.81i 48.72fgh 39.87h 61.32def 49.78fgh 57.45efg 67.43de 72.61cd 86.62bc 66.18de 90.30b 66.48de 107.64a 2.58 *** *** ***
Ser 47.72 39.54bcdefef 32.58bcdefg 51.98abcd 78.91a 46.47abcde 63.64ab 24.65cdefg 57.04abc 9.88fg 40.12bcdef 5.04g 32.48bcdefg 18.10efg 19.08defg 5.92 *** *** *
Glu 0.31 23.79g 19.38g 117.24bc 105.89cd 94.36cde 120.41bc 43.81fg 146.65ab 62.24ef 151.93ab 35.55fg 170.22a 71.03def 126.58bc 6.64 *** *** ***
Gly 20.91 16.39g 14.82g 46.56ef 41.97f 60.66de 48.84ef 85.69bc 60.17de 89.37abc 73.80cd 102.58a 79.59bc 90.75ab 88.40abc 2.80 *** *** **
Ala 89.09 82.10 87.09 128.16 117.25 138.45 129.62 128.48 118.31 134.50 126.74 151.79 135.85 120.48 134.38 4.63 *** ns ns
Val 8.75 10.65f 24.19f 74.86d 58.66e 87.55d 73.22de 116.22bc 84.77d 127.97ab 106.06c 143.06a 115.39bc 121.21bc 121.10bc 2.84 *** *** ***
Met 0.26 11.86e 16.23e 57.82bcd 50.56cd 51.23cd 41.81d 63.17bc 42.17d 72.88ab 56.60bcd 87.48a 63.30bc 65.23bc 66.69bc 3.45 *** *** **
Ile 5.00 18.69g 28.56fg 50.29e 40.02ef 57.29de 46.68ef 90.51bc 59.22de 99.95ab 74.39cd 117.17a 84.69bc 97.68b 94.09b 3.41 *** *** ***
Leu 14.84 27.73g 42.57g 113.45de 82.50f 117.51de 105.57ef 157.58bc 117.62de 163.47ab 135.05cd 185.48a 147.00bc 161.56ab 163.16ab 4.32 *** *** ***
Tyr 4.89 33.37b 61.74a 18.42bc 2.70c 11.89c 33.01b 2.80c 14.38bc 4.39c 22.25bc 11.19c 19.30bc 3.68c 3.32c 3.69 *** *** ***
Phe 8.67 32.78de 54.54cde 89.25bc 25.29e 81.17bc 74.25bcd 101.68ab 73.69bcd 110.71ab 98.49abc 134.58a 106.29ab 102.52ab 105.05ab 8.01 *** ** **
His 0.00 1.36f 36.96de 30.30de 8.90ef 65.71bc 41.59cd 71.59b 42.09cd 90.91ab 70.97b 117.96a 74.72b 78.78b 74.04b 5.10 *** *** ***
Orn 0.00 0.00b 0.00b 0.00b 8.54b 4.75b 46.44a 0.00b 10.29b 0.00b 17.26b 0.00b 5.60b 0.00b 0.27b 3.53 *** *** ***
Lys 12.61 45.13 56.55 48.37 39.24 47.79 93.18 90.68 111.56 127.95 150.91 124.96 157.45 114.30 168.03 11.84 *** ** ns
Arg 15.86 26.54 24.10 9.47 4.52 7.79 9.33 5.37 8.61 17.30 17.32 15.40 12.26 8.52 7.12 1.56 *** ns ns
Pro 8.75 7.08h 12.26fgh 18.13fgh 9.65gh 25.18efg 27.93ef 51.67cd 35.42de 72.19ab 49.28cd 76.78a 55.60bc 65.39abc 62.90abc 3.02 *** *** **
Total AA 347.3 529.7 637.1 1003.8 830.6 1093.1 1127.3 1213.4 1181.0 1395.7 1413.0 1521.7 1487.9 1335.4 1491.1 47.9 *** ns ns
Flavours1:
Sweet 183.7 190.7 168.6 293.6 287.7 332.1 319.8 348.0 338.4 378.5 376.6 402.4 393.8 361.2 412.4 14.4 *** ns ns
Bitter 37.5 101.7h 166.1gh 385.7de 257.0fg 394.8de 341.5ef 529.2bc 377.5de 575.0ab 470.6cd 667.8a 516.7bc 548.2bc 550.1bc 16.6 *** *** ***
Acid 7.6 39.1h 56.4h 160.0efg 128.9g 203.3cde 111.3def 147.2fg 212.5bcd 200.1de 251.1ab 197.6de 278.8a 209.7bcd 247.1abc 8.01 *** *** ***
Aged 24.8 64.6 95.6 79.3 132.6 102.9 145.5 125.2 149.6 179.3 201.4 180.3 210.7 177.9 217.8 12.7 *** ** ns
‘‘In same row, means with different letters are significantly different”.
RT – ripening time effect; S – salt effect. ns = not statistically significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.1Bitter flavour = R of leucine, valine, isoleucine, methionine and phenylalanine. Sweet flavour = R of alanine, glycine, threonine, serine and proline. Acid flavour = R of glutamic acid, aspartic acid and histidine. Aged flavour = Rof lysine, tyrosine and aspartic acid.
L.C
.R
oseiro
eta
l./Mea
tS
cience
79
(2
00
8)
78
4–
79
4791
792 L.C. Roseiro et al. / Meat Science 79 (2008) 784–794
Despite the inhibitory effect of salt on aminopeptidaseactivity reported in previous studies (Toldra et al., 1993,1993), it is also stated that these enzymes are still quiteactive at aw values found in ‘‘Painho de Portalegre”. Theexpected NaCl inhibition could in the present study becounterbalanced by variations in drying/smoking tempera-ture, which is frequently observed in traditional processingmethods. Once the drying/smoking phase is concluded(S3), total FAA level was almost double that found in sea-soned raw materials (3%–529.7 mg/100 g vs 1213.4 mg/100 g; 6%–637.1 mg/100 g vs 1181.0 mg/100 g). This freeamino acid accumulation still rose significantly in S4 andS5 samples, reaching +15% and +9% in 3% ‘‘Painho de
Portalegre” and +19% and +5.3% in 6% ‘‘Painho de Por-talegre”. Maintaining the ripening/ageing process up to180 days (S6), stabilized total FAA content in 6% ‘‘Painho
de Portalegre” and showed a slight decrease in 3% ‘‘Painho
de Portalegre”. Mateo, Dominguez, Aguirrezabal, andZumalacarregui (1996); Beriain, Lizaso, and Chasco(2000) reported similar trends and concentrations in tradi-tional ‘‘chorizo” and ‘‘salchichon”.
Among the 16 free amino acids detected in raw batters(R0) (Table 4), alanine and taurine were highest(89.09 mg/100 g and 85.13 mg/100 g on DM, respectively),followed by serine, glycine, treonine, arginine, leucine,and lysine ranging between 47.72 mg/100 g and12.61 mg/100 g on DM. The remaining amino acids con-centrations were below 10 mg/100 g DM, according tothe following decreasing order: valine, proline, phenylala-nine, aspartic acid, isoleucine, tyrosine and methionine.This FAA profile differed from those reported by Mateoet al. (1996) in ‘‘chorizo” (glutamic acid, taurine, alanine,lysine) and Beriain et al. (2000) in ‘‘salchichon” (glutamicacid, cisteine, alanine, taurine). The relative high concen-tration of taurine in raw material mixes (�24% of totalFAA), a non-protein amino acid, was also a characteristicobserved by De Masi (1990) in fermented and non-fer-mented sausages. The decrease of serine concentration in3% ‘‘Painho de Portalegre” after the processing period(S3) and of arginine in both formulations during the fer-mentation stage, were the only exceptions to the generalincreasing trend observed for single FAA concentrationsduring the ripening/ageing of products. During storage,the individual FAA accumulation rates slightly differedbetween 3% and 6% ‘‘Painho de Portalegre”. At 90 daysof ripening (commonly the period before consumption),quantitatively the most important amino acids were, indecreasing order, methionine, glutamic acid, histidine, iso-leucine, valine, phenylalanine, leucine and aspartic acid in3% ‘‘Painho de Portalegre” whereas in 6% ‘‘Painho de
Portalegre” the profile changed to glutamic acid, methio-nine, isoleucine, valine, phenylalanine, lysine, leucine andproline. Such differences indicate the possibility of fla-vour/taste impacts on the final products. Since specificamino acid groups may have an influence exceeding theindividual effects on those sensorial attributes (Henriksen& Stahnke, 1997), the concentration of amino acids asso-
ciated with sweet, bitter and acid/sour tastes and aged fla-vour have been calculated in both formulations (Table 4).In spite of the salt content, S4, S5 and S6 final productsshowed similar concentrations (p > 0.05) for the aminoacid group related to sweetness. Differently, the impor-tance of bitterness taste groups was greater in 3% ‘‘Painhode Portalegre” while the acidic taste groups dominated in6% ‘‘Painho de Portalegre”. Bitterness of dry fermentedsausages has been related to hydrophobicity of proteinbreakdown products and, in fact, 3% ‘‘Painho de Portal-
egre” had systematically higher proportions of non-polarfree amino acids than its counterpart. However, this gapdecreased during ripening (+40.2% in S3, +29% in S5and was almost non-existent in S6 samples). Concerningthe acid taste amino acid group, the prevalence in 6%‘‘Painho de Portalegre” is relevant and resulted from theconsiderable increase in glutamic acid level (from0.31 mg/100 g DM in raw material to 151.93 mg/100 gDM after 90 days of ripening), over twice the amountfound in the 3% formulation (62.24 mg/100 g DM). Asmentioned for bitterness, differences in acidic characteris-tics between samples ripened for 180 days were not signif-icant. Six percent ‘‘Painho de Portalegre” also had higherconcentrations of the amino acid group associated withaged flavour, with the difference slightly increasing withripening time, basically caused by more tyrosine andlysine but less aspartic acid than 3% ‘‘Painho de
Portalegre”.Trends reported could be due to distinct performances
of muscle and microbial aminopeptidases under the pro-cessing conditions applied and to different patterns ofFAA utilization for volatile compound formation (Buscail-hon, Monin, Cornet, & Bousset, 1994). Confirming Toldraet al. (1992), aminopeptidases showed reduced activity withthe elongation of the ripening period, probably due to theirown proteolysis and to the presence of an higher salt con-centration (Buscailhon et al., 1994).
4. Conclusions
Three percent and six percent salted ‘‘Painho de Portal-
egre” dry fermented/ripened sausages differed in importantaspects of proteolysis, which could be determinant fordefining the taste and flavour attributes. The ripeningobserved after processing played, as expected, a key influ-ence on the rate and extent of prevalent metabolicmechanisms.
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