Efficacies of Garlic and L.

sakei in Wine-Based
Marinades for Controlling Listeria monocytogenes
and Salmonella spp. in Chouriço de Vinho, a Dry
Sausage Made from Wine-Marinated Pork
Marı́a Belén Linares, Marı́a Dolores Garrido, Conceição Martins, and Luis Patarata

Abstract: Chouriço de vinho is made from roughly minced (10 to 30 mm) pork and fat, seasoned with a marinade made
from wine, salt, garlic, and other facultative seasonings used according to the recipe of each producer. The batter is
maintained at 4 to 7 ˚C for 24 to 48 h. It is then stuffed into natural thin pork gut, cold smoked and matured at a low
temperature for 1 to 4 wk. The effect of garlic used in wine-based marinade and a starter culture of indigenous Lactobacillus
sakei on the behavior of Listeria monocytogenes and Salmonella spp. in the processing of chouriço was investigated. The
garlic (as powder and fresh juice) was found to contribute (P < 0.05) to the control of both pathogens in broth. Garlic
dose, as tested within the usual limits used for seasoning, did not impact the reduction of pathogens. Garlic-wine-based
marinade and a starter culture of indigenous L. sakei contribute to controlling L. monocytogenes and Salmonella spp. in
the processing of chouriço. Their presence was responsible for the loss of viability of L. monocytogenes and Salmonella spp.
following 5 d of drying, even sooner than situations where no garlic was used. The results of the present work show that
the use of a wine-based marinade with garlic has an important role in ensuring the safety of the product.

M: Food Microbiology
Keywords: foodborne pathogens, garlic, sausage, wine marinade

& Safety
Practical Application: Demonstration that traditional meat preservation techniques using natural ingredients, namely,
by the use of wine-based marinades with garlic, are useful in the control of foodborne pathogens.

Introduction
Historically, the tradition of wine making in Portugal has been
associated with the production of sausages for which wine serves
as the main seasoning. The wine, usually red, is the base of a
characteristic marinade called vinha d’alhos (literally, “vineyard of
garlics”), which is made with salt, garlic, and other facultative
seasonings used according to the recipe of each producer. This
marinade is used both in the cooking of pork and the preparation
of dry pork sausages (Patarata and others 1998). Of the sausages
that use the vinha d’alhos marinade, chouriço de vinho (literally, “wine
chourizo”) is one of the most important, as it represents a prod-
uct with a high production share. Chouriço de vinho (henceforth
referred to as chouriço) is generally made from roughly minced (10
to 30 mm) pork and fat, seasoned with the wine marinade previ-
ously described. The batter is maintained at a low temperature (4
to 7 ˚C) for 24 to 48 h to allow the penetration of marinade in the
meat. It is then stuffed into natural thin pork gut, allowed to drain
the excess of marinade and to slightly dry the surface to increase
the smoke adhesion. It is than cold smoked and matured at a low
temperature for 1 to 4 wk (Colaço-do-Rosário and others 2000).
MS 20121197 Submitted 8/30/2012, Accepted 1/30/2013. Authors Linares and
Garrido are with Dept. of Food Technology, Human Nutrition and Bromathology,
Veterinary Faculty, Univ. of Murcia, Espinardo, 30071, Murcia, Spain. Authors
Martins and Patarata are with Centre of Studies in Animal and Veterinary Science
(CECAV), Univ. de Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal.
Direct inquiries to author Patarata (E-mail: blinares@um.es).
Previous studies have examined the effects that marinating meat
has on the sensory proprieties of fresh meat designated for cooking
(Oreskovich and others 1992; Onenc and others 2004) or con-
trolling spoilage microflora (Friedman and others 2007; Kargiotou
and others 2011). These works have chiefly addressed marinades
prepared with food additives for pH regulation. The inhibitory ef-
fect of garlic on foodborne pathogens is well documented (Johnson
and Vaughn 1969; Kumar and Berwal 1998), particularly in light
of the abundance of studies on its essential oil (Ross and others
2001) or the isolated chemical compounds in garlic (Ankri and
Mirelman 1999; Miron and others 2000). It is generally accepted
that the inhibitory effect that garlic or its essential oil has on sev-
eral bacteria is mainly attributed to allicin (diallyl thiosulfinate), a
substance that can inhibit enzymes containing sulfhydryl groups
(Corzo-Martı́nez and others 2007). Kumar and Berwal (1998)
and Shobana and others (2009) showed that freshly grounded
garlic cloves or aqueous extracts of garlic had inhibitory activity
against several enteric pathogens, Staphylococcus aureus and Listeria
monocytogenes.
The possible ways in which the inhibitory constituents of garlic
interact with the components of chouriço are not completely un-
derstood at present, nor is the manner in which chouriço’s safety
is achieved by the combination of several hurdles to the survival
and multiplication of pathogens, namely, reduced water activity,
pH, smoking, presence of lactic acid bacteria (LAB), and chemi-
cal preservatives. How well the results obtained with garlic oil or
specific compounds in culture media may be extrapolated to the
reality of processing of dry-cured sausages is also not completely



C 2013 Institute of Food Technologists
R

doi: 10.1111/1750-3841.12094 Vol. 78, Nr. 5, 2013 r Journal of Food Science M719
Further reproduction without permission is prohibited
 Wine and garlic marinade sausage safety . . .
Table 1–Strains used in this study.
Species Strain
Pathogens
L. monocytogenes ATCC 35152
L. monocytogenes EDS-B-LM02; EDS-B-LM05
L. monocytogenes EDS-E-LM02
Salmonella enterica subsp. enterica ATCC 49214
Salmonella spp. MPI-B-07
Salmonella spp. MPI-E-92; EDS-E-S02
LAB
L. sakei ATCC 15521
L. sakei Lch05; Lch20; Lch22; Lch36; Lch43; Lch45; Lch49;
Lch58; Lch76; Lch77; Lch78; Lch82; Lch94
ATCC = American type culture collection.
understood. The concept of hurdle technology (Leisnter 1992) is
based on the principle that any disturbance to pathogen home-
ostasis aids in its control. Thus, even if the isolated effect of garlic
in controlling pathogens in chouriço can be minor, its contribution
to the overall control, obtained by the combination of different
hurdles, is worthy of research.
The contribution of LAB to the safety of meat products is well
established (Hugas and others 1995). The use of LAB in starter
cultures obtained from naturally fermented products similar to
those where its use is intended is potentially advantageous, as
M: Food Microbiology
it is expected that those LAB have good adaptation ability and
successfully dominate the sausage microflora (Ammor and others
& Safety
2006).
Information about the use of wine-based marinades with garlic
in the preparation of dry fermented sausages is scarce, particularly
regarding its effect on foodborne pathogens and LAB used as
starter cultures. The aim of this work was to evaluate the effect
of garlic used in wine-based marinade and a starter culture of
indigenous Lactobacillus sakei on the behavior of L. monocytogenes
and Salmonella spp. in the processing of chouriço.
Material and Methods
Organisms and growth conditions
Three L. monocytogenes, 3 Salmonella spp., and 13 L. sakei strains
isolated either from chouriço or from the environment of its pro-
duction were used in this study and are listed in Table 1. For
each species, a strain from ATCC was also included. The strains of
pathogens, maintained at −18 ˚C, were subcultured twice in Brain
Heart Infusion (BHI, Biokar, Beauvais, France) and incubated for
24 h before use. L. monocytogenes was incubated at 30 ˚C and
Salmonella spp. at 37 ˚C. Cultures for inoculation were grown in-
dividually overnight in 30 mL of BHI, harvested by centrifugation,
washed twice, and resuspended in 0.85% NaCl. A cocktail of 4
strains of each pathogen was prepared to achieve the level of inocu-
lation desired, starting with a suspension with a turbidity similar to
the Macfarland standard number 1 (Biomerrieux, Marcy-l’Etoile,
France). Preparation of L. sakei strains was similar to the procedure
indicated for the pathogens but using Man-Rogosa-Sharpe (MRS,
Biokar) and incubation at 30 ˚C.
Antagonistic activity of L. sakei against foodborne
pathogens
The research for the antagonistic activity of LAB against
pathogens was conducted using the spot on agar test and well
diffusion, essentially as described by Ammor and others (2006),
M720 Journal of Food Science r Vol. 78, Nr. 5, 2013
Source/reference
ATCC
chouriço batter; laboratory collection
Environment of chouriço preparation; laboratory collection
ATCC
chouriço batter; laboratory collection
Environment of chouriço preparation; laboratory collection
ATCC
Traditional fermented sausages; laboratory collection
and included 13 strains of L. sakei tested individually against a
cocktail of 4 strains of L. monocytogenes or Salmonella spp.
Growth in adverse conditions
Two different broths were inoculated, one of which was MRS,
used as control (C), and the other of which simulated some of
the adverse conditions prevailing in chouriço: MRS supplemented
with 5% red wine (alcohol 11%, pH 3.8), 1% garlic juice, 2%
salt, 150 mg/kg KNO3 , and 150 mg/kg NaNO2 (WGSN). Fifty
milliliters of culture medium (in triplicate) were inoculated with
0.1 mL of cellular suspension (approximately 8 log CFU/mL) and
incubated at both 7 and 15 ˚C for 5 d. After 12 h of incubation
(and at every 24 h subsequently), the optical density of the culture
was measured (600 nm, 10 mm, Jasco-V-53, Essex, U.K.).
Antibacterial activity of garlic against pathogens on
culture media
Because the garlic used by chouriço manufacturers can be crushed
or mashed fresh cloves, dried granules or powder, 2 types of garlic
were tested in the present study: (1) freshly prepared juice obtained
from peeled and washed garlic cloves using a domestic centrifugal
juicer (Kenwood Chef-AT641, Maia, Portugal), which substituted
for the crushing or mashing procedures that were difficult to stan-
dardize; and (2) a 10% suspension of commercial garlic powder
(Ducros-Margão, Vila-Franca-de-Xira, Portugal) in water.
To study the eventually lethal effect of garlic on pathogens, a
study on broth culture medium was conducted (Dadadioglu and
Evrendilek 2004). As garlic is generally used in combination with
red wine in seasoning chouriço, the combined effect of garlic and
wine was tested. Considering that ethanol is the major constituent
of wine with potential antimicrobial proprieties, the effect of gar-
lic was also tested in a 10% suspension of ethanol (pH 6.4). BHI
was inoculated with 0.1 mL of each pathogen cocktail to obtain
a concentration of viable cells between 5 and 6 log CFU/g. An
experimental design was prepared for the garlic type (powder or
juice), garlic level (control, 0.5%, 1%, and 2%) and the excipient
(wine or ethanol 10%). The levels of garlic and wine chosen in
this work are between the limits usually used for sensory purposes.
Garlic is typically used in the preparation of marinades for chouriço
in a concentration around 1%. Approximately 0.1 mL of the gar-
lic preparation was added to 1 mL of inoculated BHI. The same
amount of wine or ethanol solution was added to control samples.
Enumeration of survivors was made possible by plating 10-fold
dilutions in Oxford agar (OA, Biokar) (30 ˚C, 48 h) and xylose ly-
sine desoxycolate (XLD, Biokar) (37 ˚C, 24 h) for L. monocytogenes
and Salmonella spp., respectively. The experiment was conducted
in triplicate.
Wine and garlic marinade sausage safety . . .

The effect of the addition of garlic and selected L. sakei on Table 2–Inhibitory activity of L. sakei strains exhibiting antibac-
terial activity.
microflora behavior in chouriço
Chouriço manufacturing and inoculation. The experiment Indicator organisms
was designed to test the influence of: (1) the addition of 1% gar- L. monocytogenes Salmonella spp.
lic juice (absent/present) and (2) an L. sakei starter culture (ab- L. sakei
sent/present). The experiment was conducted in quadruplicate. strains SOA WD WDN SOA WD WDN
All 4 batches were manufactured with ground pork belly (15 mm; Lch05 +a − − − − −
Mainca, Barcelona, Spain), 5% regional red wine (11% ethanol, Lch20 +++ + + + − −
Lch45 +++ + + ++ − −
pH 3.8), 1.7% salt and 125 mg/kg NaNO2 and 125 mg/kg KNO3
Lch49 + − − − − −
(Giulini-Chemie, Ludwigshafen, Germany), and the variable in- Lch58 ++ + − − − −
gredients of the batch (percentages are based on the batch weigh). Lch76 ++ − − − − −
A suspension of L. sakei was added to reach an initial concen- Lch77 + − − − − −
tration of 6 log CFU/g. Each batch was divided in 2 portions, SOA = Spot on agar; WD = Well Diffusion; WDN = Well diffusion neutralized
one inoculated with the cocktail of Salmonella spp. and the other supernatant.
a
Distance between the edge of the spot and the limit of the inhibition zone: – absence of
with L. monocytogenes to obtain an initial value between 2 and 3 inhibition; + < 5 mm; ++ 5–10 mm; +++ > 10 mm.
log CFU/g, to simulate a realistic level of contamination of meat.
Once the preservation of this chouriço is based in a combination of
several hurdles, we used a low initial contamination to avoid the 10%) against pathogens on culture media and the effect of the
possibility that hurdles to growth may be overwhelmed due to the addition of garlic (absent/1%) and L. sakei starter culture (ab-
inoculum size, leading to the conclusion that the formulation is sent/present) on the count of Salmonella spp. in chouriço was an-
not stable (Notermans and others 1993). After mixing (10 min; alyzed by factorial ANOVA. The Tukey HSD test was used to
Mainca), the batter was left to rest for 24 h at 7 ˚C before stuffing. determine the significant differences (P < 0.05) between group
The chouriços were then stuffed into natural thin pork gut, and means. Once the counts of L. monocytogenes after smoking were
tied in a ring (each sausage weighed between 250 to 300 g). The below the DL, its presence in 10 g of chouriço after enrichment

M: Food Microbiology
chouriços were left to slightly dry the surface for 4 h (7 ˚C, 85% was screened. The chi-squared test was applied to evaluate the
relative humidity) and were smoked for 3 h in a smoking chamber relationship between the presence/absence of garlic and the pres-

& Safety
(Begarat, Thermaxs-100EC, Berlin, Germany) with smoke from ence/absence of L monocytogenes in 10 g of chouriço (Statistica 7.0,
beech wood scraps. The maximum temperature reached during StatSoft Inc., Tulsa, Okla., U.S.A.).
smoking was 35 ˚C. The chouriços were then dried at 15 ˚C, 85%
RH (Aralab-Fitoclima, Rio-de-Mouro, Portugal) for 3 wk. Sam-
ples were collected for analysis 24 h after the preparation of the Results and Discussion
batter (at stuffing), after smoking and at 5, 12, and 21 d of drying. Antagonistic activity of L. sakei against foodborne
Microbial analysis pathogens
Ten grams of chouriço was collected from each sausage and diluted The results of the antagonistic activity of strains of L. sakei
with 40 mL of buffered peptone water (BPW, Biokar). The sample against a cocktail of 4 strains of L. monocytogenes or Salmonella
was homogenized for 2 min in a Stomacher (IUL, Barcelona). The spp. are presented in Table 2. Of the 14 strains of L. sakei tested,
Salmonella spp. counts were obtained after incubation on XLD a total of 7 were shown to produce inhibition zones against L.
at 37 ˚C for 24 or 48 h and the L. monocytogenes counts were monocytogenes using the spot on agar test. Against Salmonella spp.,
obtained after incubation on OA at 30 ˚C for 48 h. On the batter only 2 strains (LCh20 and LCh45) were able to show inhibition.
and smoking sampling, 0.1 mL of the first dilution was plated When the antagonism was tested using the sterile supernatant, the
on the surface of the appropriate medium in 90 mm Petri dishes inhibition of L. monocytogenes was achieved only with 3 strains,
(detection limit – DL- 0.9 CFU/g). To obtain a lower DL, on one of which was associated with acid, given that the antagonism
the 5th d and thereafter, 0.5 mL of the first dilution was plated was not observed when the pH of the supernatant was adjusted to
on the surface of 150 mm Petri dishes (DL 1.6 CFU/g). Samples pH 6.5. The strain Lch45 had the broadest antagonism and was
with counts below the DL at 24 h of incubation were screened selected for inoculating the chouriço.
for the presence of the pathogen in 10 g; presumptive colonies in
selective media were confirmed by the PCR basically as described Growth of L. sakei in adverse conditions
by Talon and others (2007). LAB counts were obtained on MRS The growth of the 14 strains of L. sakei tested in adverse con-
(30 ˚C, 72 h). ditions was studied. The results presented here relate only to
Physicochemical analysis the 2 strains that showed a more interesting antagonistic activity
The pH was measured (in triplı́cate) directly in the sausages (Figure 1). The pattern of growth was similar for both strains. The
(or batter) using a pH meter (Crison, Barcelona) with a penetra- effect of supplementation of culture broth with wine, garlic, salt,
tion probe (Mettler-Toledo, Giesen, Germany). The water activity sodium nitrite, and potassium nitrate had no discernible effect on
was measured (in duplı́cate) in a Hygroscope DT apparatus with the growth of the strains tested. The lower incubation temperature
aWA40 cell (Rotronic, Zurich, Switzerland) maintained at 20 ± delayed the growth slightly. The 2 temperatures selected are those
2 ˚C. The results are presented as the mean and standard deviation used in the 2 stages of chouriço preparation: 7 ˚C, the temperature
of the 4 repetitions of the experiment. of marination, and 15 ˚C, the temperature of drying. These strains
of L. sakei were isolated from a sausage similar to the chouriço stud-
Data analysis ied in this work, making them good candidates for improving the
The antibacterial activity of garlic (type powder or juice and microbiological safety of chouriço, as they are well adapted to the
level control, 0.5%, 1%, 2%) and the excipient (wine or ethanol conditions of the product and are consequently more competitive.

Vol. 78, Nr. 5, 2013 r Journal of Food Science M721

 Wine and garlic marinade sausage safety . . .

Table 3–Counts (log CFU/mL) of L. monocytogenes and Salmonella spp. after the addition of garlic suspended in red wine or ethanol
(10% in BHI).

Garlic powder Garlic juice

Control
Pathogen Excipient (without garlic) 0.5% 1% 2% 0.5% 1% 2%
L. monocytogenes
Wine 5.71 ± 0.41d1 4.31 ± 0.13ab 4.39 ± 0.10ab 4.23 ± 0.10a 4.72 ± 0.24abc 4.44 ± 0.66ab 4.20 ± 0.10a
Ethanol 5.53 ± 0.09cd 4.64 ± 0.11ab 4.28 ± 0.16ab 4.18 ± 0.33a 5.10 ± 0.50bcd 4.44 ± 0.08ab 4.44 ± 0.18ab
Salmonella spp.
Wine 5.74 ± 0.21c 5.23 ± 0.07abc 4.90 ± 0.23ab 4.37 ± 0.22a 4.99 ± 0.26abc 4.80 ± 0.19ab 4.78 ± 0.32ab
Ethanol 5.76 ± 0.18c 4.99 ± 0.26abc 4.63 ± 0.72ab 4.70 ± 0.18ab 5.12 ± 0.11abc 4.64 ± 0.25ab 4.80 ± 0.07ab
a,b,c,d
Means in the pair of rows of each pathogen without the same letters are significantly different (P < 0.05).

Antibacterial activity of garlic against pathogens on
culture media
The bactericidal effect of garlic juice and garlic powder was
tested in broth supplemented with 3 levels of garlic falling within
the typical limits for the seasoning of chouriço (0.5%, 1%, and 2%)
and a negative control that lacked the addition of garlic. The
garlic was added with red wine in a proportion similar to that
used in the seasoning of chouriço. To rule out the antimicrobial
effect of wine itself, garlic was also tested in a 10% solution of
ethanol, approximating the ethanol levels in the wine used in
the manufacturing of chouriço. The counts of L. monocytogenes and
M: Food Microbiology
inhibition between different microorganisms may be due to differ-
ing compositions of bacterial membranes and their permeability.
Considering that the expected effect of active compounds of gar-
lic is observed inside the target cell, inhibiting thiol-containing
enzymes by the rapid reaction of thiosulfinates and thiol groups
(Ankri and Mirelman 1999), any factor that can increase the per-
meability of the target cell to the active compounds of garlic will
result in higher inhibition. The structural aspects of the target
pathogen might have an important role in its response to the in-
hibitory effect. The outer membrane of the gram-negative cell
wall could constitute an important permeability barrier to antimi-

Salmonella spp. survivors are presented in Table 3. The addition of crobial compounds (Miron and others 2000). Additionally, as the
garlic, whether as freshly prepared juice or as powder suspension, garlic tested in the present work was added to wine or an ethanol
& Safety

resulted in lower counts for both pathogens. The use of wine or an
ethanol solution resulted generally in similar (P > 0.05) survival
rates for both pathogens.
When added with wine, the effect of garlic on L. monocytogenes
resulted in a significant reduction (P < 0.05) in survivors, with
results between 1 and 1.5 log CFU/mL. While higher concentra-
tions of garlic resulted in still lower counts, the differences were
not significant. When the effect of garlic was tested in an ethanol
solution, the pattern of inhibition observed was generally similar.
The effect observed on Salmonella spp. was slightly weaker than
that observed for L. monocytogenes. The highest absolute reduc-
tion in the counts of Salmonella spp. achieved with 1% or 2% or
garlic was approximately 1 log CFU/mL. In the present work,
Salmonella spp. were more resistant to garlic than L. monocytogenes.
Similar results were observed by Ross and others (2001), who also
observed that higher doses of garlic oil were needed to inhibit
Salmonella spp. than that required for L. monocytogenes. However,
when these authors tested the resistance with garlic powder, the
inhibitory dose was similar for both pathogens. The differences in
solution, the effect of ethanol in the permeabilization of the mem-
brane (Barker and Park 2001) should be considered as a potentially
synergistic effect in the inhibition of the pathogen.
Although the isolated effect of garlic in controlling L. mono-
cytogenes and Salmonella spp. in chouriço can be considered minor,
given that the inhibition observed in broth was between 1 and
1.5 CFU/mL, the contribution made by garlic to the overall con-
trol of these pathogens through the combination of the presen-
tation of a number of different factors (reduced water activity,
competitive microflora, chemical preservatives and smoke com-
pounds, among others) should not be neglected, even if the safety
and longer shelf life of chouriço are achieved by a combination of
factors.
The effect of garlic and L. sakei on pathogen survival
during the processing of chouriço
Table 4 presents the results of the survival of L. monocytogenes
and Salmonella spp. in chouriço prepared both with and without the
addition of garlic or the introduction of a L. sakei starter culture

2.5 Figure 1–Growth of L. sakei Lch20 and Lch45

Arbitrary units (DO 600nm, 10mm)

in MRS without glucose (C) or in MRS

suplemented with 7.5% red wine, 1% salt,
2 Lch20 C-7ºC 1% garlic juice, 150 mg/kg NaNO3 , and
150 mg/kg KNO3 (WSGN) at 7 and 15 ˚C.
Lch45 C-7ºC
1.5 Lch20 WSGN-7ºC
Lch45 WSGN-7ºC
1 Lch20 C-15ºC
Lch45 C-15ºC
0.5 Lch20 WSGN-15ºC
Lch45 WSGN-15ºC
0
12 h 24 h 48 h 72 h 96 h 120 h

M722 Journal of Food Science r Vol. 78, Nr. 5, 2013

Wine and garlic marinade sausage safety . . .

Table 4–The effect of garlic and L. sakei on the survival of L. monocytogenes and Salmonella spp. in chouriço. The L. monocytogenes
results are expressed as the number of samples (percentage) in which the pathogen was detected in 10 g; Salmonella spp. results
are expressed as log CFU/g (expressed as mean±standard deviation).

Control Garlic (1%) SignificanceA

Microorganism
Processing phase No starter L. sakei No starter L. sakei Garlic L. sakei Int.
L. monocytogenes
Batter 4 (100%) 4 (100%) 4 (100%) 4 (100%) ns ns
After smoking 3 (75%) 2 (50%) 0 0 0.031 ns
5 d of drying B 3 (75%) 1 (25%) 0 0 ns ns
Salmonella spp.
Batter 2.78 ± 0.19 2.77 ± 0.16 2.57 ± 0.14 2.37 ± 0.12 0.002 ns ns
After smoking 2.56 ± 0.34 1.97 ± 0.98 1.03 ± 0.32 0.85 ± 0.99 <0.001 ns ns
5 d of drying 2.15 ± 0.41b 0.00 ± 0.00a 0.65 ± 0.79a 0.00 ± 0.00a 0.006 <0.001 0.006
12 d of drying B 0.73 ± 0.85 <DL <DL <DL ns ns ns
A
Comparison of occurrence of L. monocytogenes was performed through the chi-square test; Comparison of Salmonella spp. counts was made by ANOVA.
B
After 5 d of drying (for L. monocytogenes) or 12 d of drying (for Salmonella spp.), all of the samples were below the detection limit, and the pathogen was not detected in 10 g of
chouriço. DL = Detection limit; ns = not significant.

Table 5–The effect of garlic and L. sakei on LAB counts and on the pH of chouriço (expressed as mean±standard deviation).

Control Garlic (1%) SignificanceA

Microorganism
Processing phase No starter L. sakei No starter L. sakei Garlic L. sakei Int.
LAB
Batter 2.54 ± 0.09c 6.36 ± 0.02a 2.12 ± 0.08b 6.37 ± 0.11a 0.003 <0.001 0.002
After smoking 4.22 ± 0.14c 7.47 ± 0.01a 3.54 ± 0.24b 7.41 ± 0.08a 0.002 <0.001 0.006
5 d of drying 4.98 ± 0.28c 8.41 ± 0.07a 4.36 ± 0.06b 8.08 ± 0.21a 0.002 <0.001 ns

M: Food Microbiology
12 d of drying 7.02 ± 0.08c 8.81 ± 0.17a 7.47 ± 0.06b 8.86 ± 0.05a 0.003 <0.001 0.008
21 d of drying 7.61 ± 0.24a 8.01 ± 0.22ab 7.67 ± 0.32ab 8.23 ± 0.09b ns 0.008 ns

& Safety
pH
Batter 5.76 ± 0.11 5.69 ± 0.03 5.69 ± 0.01 5.66 ± 0.23 ns ns ns
After smoking 4.84 ± 0.01b 4.86 ± 0.03b 4.85 ± 0.06b 4.72 ± 0.02a 0.010 0.023 0.005
5 d of drying 5.19 ± 0.02b 5.14 ± 0.03a 5.18 ± 0.01ab 5.08 ± 0.01c 0.002 <0.001 0.036
12 d of drying 5.29 ± 0.04 5.23 ± 0.04 5.23 ± 0.05 5.18 ± 0.06 ns ns ns
21 d of drying 5.24 ± 0.04 5.18 ± 0.03 5.23 ± 0.06 5.22 ± 0.12 ns ns ns
ns = not significant.
a,b,c
Means in the pair of rows without the same letters are significantly different (P < 0.05).
A
Comparisons of occurrence of LAB and pH performed using ANOVA.

during the manufacturing process. After the preparation of the
batter, the mean count of L. monocytogenes was approximately 2
log CFU/g in all of the batches. A reduction of the pathogen
was observed during the processing, leading to counts below the
detection limit. In this case, the presence of viable L. monocytogenes
in 10 g of chouriço after enrichment was screened for. Consequently,
the results for L. monocytogenes are not presented as log CFU/g, as
is otherwise the case, but as the proportion of samples in which the
viable pathogen was present in 10 g. The statistical comparisons
were made using the chi-square test. After the smoking phase, L.
monocytogenes was not detected after enrichment in 10 g of chouriço
when garlic was added, demonstrating an association (P < 0.05)
between the presence of garlic and absence of the pathogen. From
the 12th d of drying onwards, L. monocytogenes was undetectable
in 10 g of chouriço in any experimental condition.
Salmonella spp. demonstrated a higher survival rate than L. mono-
cytogenes during the processing of chouriço. Comparison with the
initial counts of Salmonella spp. in the batter allowed for the effect
of garlic addition (P < 0.05) to be determined. Counts 0.2 to
0.4 log CFU/g lower were found in samples in which 1% garlic
was added. At this sampling point, no effect of L. sakei was de-
tected. After smoking, the samples without either garlic or L. sakei
maintained Salmonella spp. counts of 2.56 ± 0.34 log CFU/g, ap-
proximately equal to the initial count found in the batter. A slight
(P > 0.05) reduction was caused by the presence of L. sakei, de-
spite the differences in pH between samples with or without starter
culture were only around 0.1 pH units (Table 5). However, the
presence of garlic in the chouriço was responsible for a reduction in
the counts of Salmonella spp. of nearly 1.5 log CFU/g. During the
subsequent 5-d drying period, the tendency toward a reduction
of Salmonella spp. continued, particularly in samples treated with
garlic or L. sakei, where significant differences were observed. In
chouriços inoculated with L. sakei, no Salmonella spp. was detected
in 10 g. When the starter culture was absent, the count of the
Salmonella spp. was higher (P < 0.05) in samples without garlic
(2.15 ± 0.41 log CFU/g) than in those with 1% garlic (0.65 ±
0.79 log CFU/g). After 12 d of drying, Salmonella spp. was present
only in samples without either garlic or L. sakei and the pathogen
disappeared entirely during the remaining drying period and was
undetectable after 21 d of drying.
The effect of garlic present in the chouriço manufactured was
generally in accordance with that observed in broth, though L.
monocytogenes was more sensitive to its presence in chouriço than
Salmonella spp were. The more pronounced effect of garlic ob-
served after smoking might be explained by the combined im-
pact of several different factors important to the survival of the
pathogens, like competitive microflora growth and consequent
pH reduction (Table 5), smoke. The heating of the chouriços to
approximately 30 ˚C may be chiefly responsible, once it is associ-
ated with an increase in membrane fluidity (Vigil and others 2005)
that increases the potential for the active compounds of garlic to
penetrate the cells, thereby enhancing their antimicrobial effect.

Vol. 78, Nr. 5, 2013 r Journal of Food Science M723

 Wine and garlic marinade sausage safety . . .

The antagonism of L. sakei observed in the spot on agar test Acknowledgments

and well diffusion assay with the strain Lch45 was also observed This work is financially supported by PEst-OE/AGR/UI0772/
in the manufacturing of chouriço. The addition of a starter culture 2011; (CECAV/UTAD) – 2011-2012.
resulted in a lower occurrence of L. monocytogenes and lower counts
of Salmonella spp.
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