LWT - Food Science and Technology 54 (2013) 51e56

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LWT - Food Science and Technology

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Probiotic strains Lactobacillus plantarum 299V and Lactobacillus

rhamnosus GG as starter cultures for fermented sausages
Raquel Rubio a, Teresa Aymerich a, Sara Bover-Cid a, M. Dolors Guàrdia b, Jacint Arnau b,
Margarita Garriga a, *
a
IRTA-Food Safety Program, Finca Camps i Armet, E-17121 Monells, Girona, Spain
b
Food Technology Program, Finca Camps i Armet, E-17121 Monells, Girona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Probiotic food products are a fast growing area. Although probiotic strains are currently used in dairy
Received 20 December 2012 products, their commercial application in fermented meat products is not yet common. The aim of this
Received in revised form study was to assess the competitiveness of two probiotic Lactobacillus strains (Lactobacillus plantarum
5 March 2013
299V and Lactobacillus rhamnosus GG) during the manufacture of Spanish fermented sausages and their
Accepted 7 May 2013
effect on the hygienic and sensory qualities of the final products. The inoculated strains were successfully
monitored by Randomly Amplified Polymorphic DNA (RAPD)-PCR. Both strains prevented the growth of
Keywords:
Enterobacteriaceae throughout the entire ripening process. L. rhamnosus GG and L. plantarum 299V at
Meat fermentation
Lactobacilli
high inoculum (ca. 107 CFU/g) produced a sharp decrease of pH values and low growth of Gram-positive
Competitiveness Catalase-positive Cocci (GCCþ), leading to a negative effect on the sensory attributes evaluated. Never-
RAPD-PCR theless, L. plantarum 299V inoculated at 105 CFU/g achieved and maintained high counts until the end of
Sensory analysis ripening and storage (ca.108 CFU/g), co-dominating (60%) with the endogenous microbiota, producing
functional sausages with a satisfactory overall sensory quality. No major differences in physico-chemical
parameters or sensory attributes were recorded when compared to spontaneously fermented sausages,
thus adding further value to this type of meat product as a probiotic vehicle.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction mainly involve the participation of Lactic Acid Bacteria (LAB),

During recent decades there has been a growing consumer de-
mand for probiotic foods, a group of functional foods which are
believed to contribute to health (Mollet & Rowland, 2002; Young,
2000). The health benefits associated with probiotic food prod-
ucts are based on the presence of selected viable strains of micro-
organisms which, when administered in adequate amounts,
improve the health of the host (FAO/WHO, 2001).
Fermented sausages are ready-to-eat products where product
safety is essentially gained by a fall in pH (4.5e5.0) and a decrease
of water activity (aw) below the growth limit of most pathogens
(<0.90), thus enabling a more efficient bacterial control within the
‘hurdle technology’ concept (Barbuti & Parolari, 2002). This process
favors the growth of the microorganisms which influence the
sensory and nutritional qualities, safety, and other key character-
istics of the final product (Martín, Colín, Aranda, Benito, & Córdoba,
2007). The fermentation and ripening of fermented sausages
* Corresponding author. Tel.: þ34 972630052; fax: þ34 972630373.
E-mail address: margarita.garriga@irta.cat (M. Garriga).
particularly Lactobacillus, and the GCCþ, mostly Staphylococcus
(Arkoudelos, Samaras, & Nychas, 1997; Lücke, 1974).
Although dairy products are the most commonly used food ve-
hicles for the delivery of probiotics, several investigations dealing
with the use of probiotics in fermented meat products to improve
their nutritional value as functional foods have been reported (De
Vuyst, Falony, & Leroy, 2008; Erkkilä, Petajä et al., 2001; Erkkilä,
Suihko, Eerola, Petäjä, & Mattila-Sandholm, 2001; Klingberg,
Axelsson, Naterstad, Elsser, & Budde, 2005; Macedo, Pflanzer,
Terra, & Freitas, 2008; Pennacchia, Vaughan, & Villani, 2006;
Rouhi, Sohrabvandi, & Mortazavian, 2013; Ruiz-Moyano, Martín,
Benito, Aranda et al., 2011; Ruiz-Moyano, Martín, Benito,
Hernández et al., 2011). In addition, the sausage matrix seems to
act as a protection, improving the survival of probiotic lactobacilli
through the gastrointestinal tract (Klingberg & Budde, 2006). As
fermented meat products are processed without heating, they could
be suitable products for assessing probiotic LAB as starter cultures
(Ammor & Mayo, 2007). Counts greater than 107 CFU/g of LAB are
usually reached in these kinds of products at the end of the process
(Benito et al., 2007; Erkkilä, Petajä et al., 2001; Erkkilä, Suihko et al.,
2001; Klingberg et al., 2005; Macedo et al., 2008). Lactobacillus

0023-6438/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.05.014
52 R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e56
species such as Lactobacillus sakei, Lactobacillus curvatus, Lactoba-
cillus plantarum and Lactobacillus casei are commonly used as starter
cultures in fermented sausages (Ammor & Mayo, 2007). Strains of
L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus as po-
tential functional starter cultures in meat products were suggested
by Macedo et al. (2008) and Rebucci et al. (2007). L. rhamnosus GG is a
well known probiotic LAB strain used to ferment dairy products
(Saxelin, 1997) and has been confirmed to be a suitable starter cul-
ture in North European acid fermented sausages (Erkkilä, Petajä
et al., 2001; Erkkilä, Suihko et al., 2001). In vitro studies carried out
by Pennacchia et al. (2006) drew attention to the suitability of
several strains of the L. plantarum-group isolated from traditional
dry-fermented sausages, for their probiotic use as starter cultures in
meat products. L. plantarum 299V (ProViva) and L. rhamnosus GG
(Gefilus, Vifit) are commercially sold as probiotics. However, in order
to choose a probiotic LAB for use as a starter culture in fermented
sausages, desirable technological, sensory and safety properties on
the final products are required. No reference about L. plantarum
299V assayed as a putative probiotic starter culture in sausage
manufacturing has been found.
The aim of the present work was to assess the suitability of two
probiotic LAB strains (L. plantarum 299V and L. rhamnosus GG) as
starter cultures for Spanish fermented sausages, by evaluating their
competitiveness during manufacturing and their effect on the hy-
gienic and sensory qualities of the final products.
2. Materials and methods
2.1. Probiotic strains
The lactobacilli strains used as probiotic starters were:
L. plantarum 299V (Probi, Sweden), L. rhamnosus GG (Valio Ltd.,
Finland) at two inoculation levels in the meat batter (105 and
107 CFU/g). Each strain was grown overnight in MRS broth (Merck,
Darmstadt, Germany) at 37  C, harvested by centrifugation at
5000 rpm for 10 min at 6  C, washed twice in saline solution (0.85%
NaCl), resuspended in saline solution and stored at 80  C with 20%
of glycerol until further use.
2.2. Sausage preparation
The fermented sausages were manufactured using 80% lean
pork and 20% pork belly, ground in a meat grinder by passing
through a 6 mm plate and mixed with the following ingredients (g/
kg): NaCl, 25; NaNO2, 0.15; KNO3, 0.15; sodium ascorbate, 0.5;
dextrose, 7; lactose, 20 and black pepper, 3.0. Five treatments of
20 kg were prepared: treatment 1 (control, without probiotic cul-
ture), treatments 2 and 3 with L. rhamnosus GG (inoculated at ca.
105 and ca. 107 CFU/g, respectively) and treatments 4 and 5 with
L. plantarum 299V (inoculated at ca. 105 and ca. 107 CFU/g, respec-
tively). After mixing, the meat batter was stuffed into 50 mm
diameter collagen casings (Colex 32, Fibran S. A., Sant Joan de les
Abadesses, Spain). The sausages were dipped into a solution of
Penicillium candidum spores (Danisco, France), fermented at 20e
22  C and 90e95% RH (until pH value ca. 5.0) and ripened at 12  C
and 75e80% RH (until aw value ca. 0.90e0.93). Ripened products
were packed into thermo sealed bags Darfresh plastic materials
(top TC201 and bottom web RSCO3X60; OTR < 2 cc/m2, 24 h, bar)
from Cryovac (Sealed Air Packaging S.L.U., Viladecans, Barcelona
Spain) and stored at 1  C for one month.
2.3. Physico-chemical parameters
For each treatment, three sausages were sampled at initial time
(stuffed batter, B), at the end of fermentation (EF) and at the end of
ripening (ER) to determine pH using a penetration electrode (Cri-
son pH meter 507, Crison Instruments S.A., Barcelona, Spain) and aw
(aw-sprint TH500, Novasina, Pfäffikon, Switzerland).
2.4. Microbiological analysis
Microbiological analyses were performed in triplicate samples
during the whole process. For each sampling time (B, EF, ER), the
casings were aseptically removed and 15 g of fuet were crumbled
and homogenized (1/10 dilution) with 0.1% Bacto Peptone (Difco
Laboratories, Detroit, MI, USA) with 0.85% NaCl (Merck) in a
Masticator Classic (IUL S.A., Barcelona, Spain) for 60 s. Serial deci-
mal dilutions were made and LAB were enumerated by plating on
de Man, Rogosa and Sharpe (MRS) agar (Merck) at 37  C for 72 h in
anaerobiosis (Anaero-Gen, Oxoid); Gram-positive catalase-positive
cocci (GCCþ) were determined by plating on Mannitol Salt Agar
(MSA, Merck) at 30  C for 48 h and Enterobacteriaceae by plating in
Violet Red Bile Dextrose Agar (VRBD, Merck) with a double layer at
30  C for 24 h. Before sensory analysis Listeria monocytogenes and
Staphylococcus aureus were determined on Chromogenic Listeria
Agar (CLA, Oxoid, Basingstoke, UK) incubated at 37  C for 48 h and
Baird-Parker agar base with egg yolk tellurite emulsion (BP, Oxoid)
at 37  C for 48 h, respectively. The presence/absence of Salmonella
was determined in 25 g according to ISO 6579 (2002). At the end of
storage LAB and GCCþ were also determined.
2.5. Strain typing
In order to monitor the inoculated probiotic strains, thirty col-
onies of LAB per treatment were randomly selected from the MRS
agar plates at each sampling time. For DNA extraction, isolated
colonies were suspended in 200 ml of 6% ChelexÒ-100 chelating ion
exchange resin (Bio-Rad, Hercules, CA, USA), heated at 100  C for
10 min, cooled on ice and centrifuged at 14,000 g for 10 min. Two
microliter of the resuspended supernatant containing the DNA was
used as a PCR template. Two random primers (Roche Molecular
Biochemicals, Indianapolis, USA) were tested in RADP-PCR analysis,
M13R2 (50 -ggaaacagctatgaccatga) and KS (50 -tcgaggtcgacggtatcg),
according to Aymerich et al. (2006) and Fulladosa et al. (2010),
respectively. Amplification products were subjected to electro-
phoresis for 150 min at 100V in 1.5% agarose (Bio-Rad) gels and
stained with 0.1 mg/ml ethidium bromide (Sigma, St. Louis, MO,
USA). Three lines of 1 kb DNA ladder (Invitrogen) were used as
molecular weight and normalization gel standards for RAPD pro-
files. The banding profiles were visualized under UV light and
digitalized by Gelprinter photodocumentation equipment (TDI,
Barcelona, Spain).
2.6. Sensory analysis
Eight trained assessors (ASTM, 1981; ISO 8586-1, 1993; ISO
8586-2, 1994) took part in the sensory analysis on 2.5 mm thick
slices. The generation and selection of the descriptors was carried
out by open discussion in three sessions according to Guàrdia,
Guerrero, Gelabert, Gou, & Arnau (2008). A non-structured 10-
point scoring scale was used, where 0 means absence or very low
intensity of the descriptor and 10 means very high intensity of the
descriptor (Amerine, Pangborn, & Roessler, 1965). Means of scores
given by the assessors for each sausage were recorded. Sensory
evaluation was undertaken in 5 sessions per product and a com-
plete block design was used (Steel & Torrie, 1983), where each taster
assessed all the treatments in each session. Samples were coded
with three-digit random numbers and were presented to the as-
sessors balancing the first order and the carry over effects according
to Macfie, Bratchell, Greenhoff, and Vallis (1989). The average score
 R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e56 53

given by the eight experts for each sample and session was recor-
ded and used in the statistical analysis.
2.7. Data analysis
Data were analyzed by means of ANOVA using the GLM proce-
dure of SAS (v9.2, SAS Institute Inc). The model for pH, aw, and
microbiological data recorded during sampling times included
treatment, time and their interaction as fixed effects. For sensory
data, the model included the treatment and the taste session as
fixed effects. Mean differences among treatments were assessed by
the post-hoc Tukey test (P < 0.05). The percentage of implantation
of a given inoculated strain was ascertained according to a sampling
plan based on the binomial distribution (Peña Sánchez de Rivera,
1986). The implantation breakpoint, defined as a percentage of
colonies that showed the same RAPD profile as the added probiotic
cultures, was set up at 70%.
3. Results and discussion
3.1. Changes in pH and water activity during ripening and storage
As shown in Table 1, the initial mean pH value of the meat batter
was 6.07. At day 6 (end of fermentation), a significant decrease of
pH was observed in all the treatments (P < 0.05), the pH values
ranging from 4.6 to 5.2.
At the end of fermentation the treatments inoculated with
L. rhamnosus GG (T2 and T3) and with the high level of L. plantarum
299V (T5) showed the highest decrease of pH (ca. 4.6) with
significantly lower values (P < 0.05) than the pH recorded for the
lot which has been inoculated with the low level of L. plantarum
299V (T4) (5.01) and the control one (5.20). Therefore, L. rhamnosus
GG showed higher acidifying ability than L. plantarum 299V. The pH
decrease contributes to the inhibition of undesirable microorgan-
isms, accelerates the reduction of nitrite to nitric oxide, affects the
flavor of the product and facilitates meat binding capacity,
improving firmness and sliceability (Lücke, 1998; Varnam &
Sutherland, 1995) and thus contributes to the safety and quality
of the sausages.
During ripening, pH was significantly different among the
treatments (P < 0.05). At the end of the process, in treatments 1 and
4 (control and L. plantarum 299V at low level) no changes were
observed in pH values compared to the values reached at the end of
fermentation (day 6), whereas in treatments 2, 3 and 5, which were
the most acid after fermentation, pH values increased slightly,
though remained at lower values than those of the spontaneously
fermented sausages (control).
The aw of the sausages decreased (P < 0.05) from an initial value
of 0.97 to 0.91e0.93 (Table 1), taking different times to reach these
values depending on the treatment. Control treatment followed by
treatment 4 required the longest ripening times (38 and 34 days,
respectively) to achieve the targeted aw values. On the other hand,
treatments showing the most marked acidification (2, 3 and 5)
resulted in a reduction of the ripening time by 47%e39% in com-
parison with the control, which could be related to the reduction of
the water-binding capacity of proteins caused by a stronger
acidification.
3.2. Bacterial growth during fermentation and ripening
Table 1 shows the results of microbial counts. Endogenous GCCþ
showed a sharp increase (P < 0.05) from ca. 3 log CFU/g to
7.14 log CFU/g at day 6 (EF) in control treatment (non inoculated
with LAB). These levels were kept constant or significantly higher at
the end of ripening (ER) (Table 1). Regarding the treatments inoc-
ulated with probiotic LAB, GCCþ counts showed an increase of ca.
1.5 log units (P < 0.05) at the end of fermentation (day 6) in the
treatments inoculated with the low level of L. rhamnosus GG and
L. plantarum 299V (treatments 2 and 4, respectively), without sig-
nificant differences between them (P > 0.05). However, in the
treatments inoculated with the high level of probiotics, the counts
remained constant compared to initial time, until the end of
fermentation (P > 0.05). At the end of ripening, the treatment
inoculated with the low inoculum of L. plantarum 299V presented a
slight increase in the counts of 0.7 logs, showing significantly
higher levels than the rest of the inoculated treatments
(5.99 log CFU/g) (P < 0.05), whereas treatment 2 remained at the
levels achieved at the end of fermentation (ca. 4.8 log CFU/g).
Treatments 3 and 5 (high levels of L. rhamnosus GG and L. plantarum
299V, respectively) showed a significant increase (P < 0.05),
reaching final counts of 4.92 and 4.32 log CFU/g, respectively. The
low levels of GCCþ in treatments 2, 3 and 5 could be explained by

Table 1
pH, aw, bacterial counts in fermented sausages during the process.

Manufacture Time Treatment

step (days)
1. Control 2. L. rhamnosus GG 3. L. rhamnosus GG 4. L. plantarum 299V 5. L. plantarum 299V
ca. 105 CFU/g ca. 107 CFU/g ca. 105 CFU/g ca. 107 CFU/g

pH B 0 6.03  0.02Ca 6.07  0.02BCa 6.05  0.02BCa 6.09  0.02ABa 6.13  0.02Aa
EF 6 5.19  0.11Ab 4.63  0.05Bc 4.58  0.03BC 5.02  0.03Ab 4.65  0.05Bc
ER 34 5.21  0.05Ab 4.85  0.03Cb 4.69  0.01Db 5.01  0.03Bb 4.79  0.02Cb
aw B 0 0.969  0.00a 0.969  0.00a 0.966  0.00a 0.967  0.00a 0.967  0.00a
EF 6 0.965  0.00Ab 0.962  0.00Bb 0.951  0.00Cb 0.964  0.00ABa 0.964  0.00ABa
ER 20 0.907  0.00Cc 0.928  0.00Ac 0.918  0.00Bc 0.908  0.00Cb 0.925  0.00ABb
LAB B 0 2.59  0.16Db 5.89  0.15Cb 7.50  0.07A 5.82  0.04Cb 7.06  0.01Bb
EF 6 8.31  0.01Aa 8.19  0.09Aa 7.48  0.41B 8.71  0.16Aa 8.51  0.02Aa
ER 38 8.84  0.23Aa 8.19  0.10BCa 7.88  0.09C 8.48  0.03Ba 8.29  0.11Ba
GCCþ B 0 3.54  0.06c 3.53  0.02b 3.62  0.09b 3.63  0.24c 3.59  0.02b
EF 6 7.14  0.18Ab 4.89  0.02Ba 3.26  0.50Cb 5.29  0.03Bb 2.78  0.36Cb
ER 23 7.63  0.14Ab 4.81  0.12Ca 4.92  0.05Ca 5.99  0.01Ba 4.32  0.20Da
Enterobacteriaceae B 0 2.50  0.08Bc 2.84  0.16Aba 2.99  0.39Aa 2.52  0.23Ba 2.49  0.10Ba
EF 6 4.75  0.08Ab 2.63  0.22Ba 1.09  0.19Db 2.71  0.01Ba 2.08  0.11Cb
ER 20 6.43  0.19Ab 1.36  0.10Cb <1.00Cb 1.83  0.26Bb <1.00Cc

Values are mean  SD of triplicates. Significant differences in rows are indicated by different capital letters and significant differences in columns are indicated by different
small letters (P < 0.05).
Bacterial counts are expressed in log CFU/g, limit of detection 1.0 log CFU/g for Enterobacteriaceae.
B ¼ stuffed batter, EF ¼ end of fermentation, ER ¼ end of ripening.
54 R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e56
the strong acidification achieved during fermentation in compari-
son with the control spontaneous fermented sausages (treatment
1). At the end of storage and for all treatments the GCCþ counts
were maintained at the levels achieved at the end of ripening.
As expected, the counts of LAB at the beginning of the process
were lower in the control treatment (endogenous microbiota) than
in those inoculated with probiotic cultures. Generally, at the end of
fermentation (day 6), the counts of LAB had increased significantly
(P < 0.05), reaching counts of 108 CFU/g which were maintained
until the end of the ripening period. According to Raccach (1992), a
rapid growth of LAB is desirable from at least two standpoints:
economic (efficiency in manufacture sausage) and public health
(control of pathogenic bacteria). At the end of the process, the
counts remained ca.108 CFU/g in all treatments, demonstrating the
competitiveness of LAB under the conditions assayed. LAB counts
and irrespective implantation of both strains were maintained till
the end of storage at 1  C at the levels achieved after ripening.
The competitiveness of the inoculated probiotic LAB strains was
specifically monitored by RAPD-PCR profiling analysis (Aymerich
et al., 2006). At day 0 and until the end of fermentation (day 6),
all the 30 isolates from each lot showed the same RAPD profiles as
the parental strain. At the end of ripening (ER), a 100% implantation
was achieved for L. rhamnosus GG at both inoculum levels (treat-
ments 2 and 3) and for L. plantarum 299V at high inoculum level
(treatment 5). In the sausages inoculated with the low concentra-
tion of L. plantarum 299V (treatment 4), with final LAB counts of
8.48 log CFU/g, 59.6% (18 patterns) of the RAPD profiles were
identified as L. plantarum 299V. Thus, the probiotic strain achieved
levels of 1.7  108 CFU/g co-dominating with other endogenous LAB
(40.4%). Although the minimum recommended daily dose of pro-
biotic bacteria is not known, it is estimated to be 109e1010 viable
cells in order to show an effect on health and a temporary coloni-
zation of the gut (by levels of 106e108 CFU/g of feces) (Työppönen,
Petäjä, & Mattila-Sandholm, 2003). Thus, for a sausage containing
108 viable cells/g, such as those of the present work, the minimum
dosage for probiotic use would be achieved by eating 10e25 g of
sausage per day, which is quite feasible and compatible with a
nutritionally balanced diet.
The RAPD-PCR study was useful in confirming the implantation of
the probiotic LAB strains in fermented sausages, verifying that this
method is adequate to discriminate lactobacilli at strain level
(Aymerich et al., 2006). The obtained results demonstrated that the
assayed probiotic cultures grew rapidly in fermented sausages and
dominated or co-dominated (L. plantarum 299V at low inoculum
level) with the endogenous LAB during the fermentation and ripening
processes, proving their suitability as probiotic starter cultures. Other
studies have ensured the suitability of probiotic strains of L. rhamnosus
and L. plantarum for use as probiotic cultures in fermented sausages
according to their adaptation to the sausage environment and their
fast growth rate and acidification. Erkkilä, Petajä et al. (2001) and
Erkkilä, Suihko et al. (2001) showed that L. rhamnosus GG,
L. rhamnosus E-97800 and L. plantarum E-98098 were suitable for use
as starter cultures in more acidic North European dry sausages.
Klingberg et al. (2005) identified L. plantarum strains originating from
the dominant non-starter LAB fermented meat products as promising
candidates for probiotic meat starter cultures suitable for the manu-
facture of Scandinavian-type fermented sausages.
Enterobacteriaceae counts were significantly different (P < 0.05)
depending on the treatment. At day 0 the counts were below
103 CFU/g (Table 1), values considered hygienically correct for raw
meat. At the end of fermentation (day 6), in the control treatment,
Enterobacteriaceae counts increased significantly (P < 0.05; to
4.75 log CFU/g) and maintained levels significantly higher than in
the LAB inoculated treatments throughout the ripening process
(P < 0.05). The probiotic inoculated sausages kept the initial
Enterobacteriaceae values or even decreased the counts in T3 (sau-
sages inoculated with high level of L. rhamnosus GG). At the end of
ripening (ER), Enterobacteriaceae counts were below 2 log CFU/g in
all the inoculated treatments, significantly lower (P < 0.05)
compared to the counts for the control lot (>6 log CFU/g). These
results confirmed the inhibitory effect of the inoculated probiotic
cultures and/or the acidification observed against Enter-
obacteriaceae, which is crucial to obtain high quality hygienic sau-
sages (Benito et al., 2007; Coppola, Marconi, Rossi, & Dellaglio, 1995;
Martín et al., 2007). High counts of these microorganisms at initial
stages of ripening are related to the production of biogenic amines
(Bover-Cid, Hernández-Jover, Miguélez-Arrizado, & Vidal-Carou,
2003) and hydrogen sulphyde odors that diminish the accept-
ability of the final product (Garriga et al., 1996). Garriga et al. (2005)
also observed that Enterobacteriaceae counts increased significantly
(P < 0.05) during the first 7 days of ripening in Spanish dry-
fermented sausages (fuet and chorizo) without starter cultures,
whereas no growth was observed in treatments with starter culture.
In the final products, the foodborne pathogens (L. monocytogenes,
S. aureus) were not detected in any treatment (values below the
detection limit <1 log CFU/g). Salmonella was not detected (absence
in 25 g) in any final product, confirming the safety of fermented
sausages according to the European safety microbiological criteria
(Regulation 2073/2005, European Commission, 2005).
3.3. Sensory evaluation
Mean scores given by the assessors for the fermented sausages
at the end of the ripening process are detailed in Table 2. Generally,
the addition of probiotic LAB cultures affected the sensory char-
acteristics of the fermented sausages, which could mainly be
related to the differences observed in pH and the lower levels of
GCCþ compared to the control treatment.
Regarding appearance, the control samples showed slightly
lower scores (P < 0.05) of visual cohesiveness which was similar in
all the inoculated lots. Control samples (treatment 1) and those
inoculated with the low inoculum level of L. plantarum 299V
(treatment 4), but co-dominating with the endogenous LAB at the
end of ripening, presented a higher intensity of red color and
brightness than T2, T3 and T5 (P < 0.05). Concerning the odor de-
scriptors, the highest values for overall odor intensity as well as for
ripened odor, were scored in control sausages and in those of T4
(P < 0.05) with slight differences. The rest of the treatments had
values significantly lower (P < 0.05) for these descriptors and
higher values of cooked odor (P < 0.05) which could be due to the
lower counts of GCCþ. These bacteria have catalase and nitrate
reductase activities which contribute to preventing oxidation in
fermented sausages. The lower counts of GCCþ, produced by the
strong decrease of pH to values below 5.0, obtained in treatments 2,
3 and 5 at the end of ripening period may have contributed to a
lower development of the characteristic red color and ripened odor
in these sausages. According to Metaxopoulos, Samelis, & Papadelli
(2001), the predominance of LAB on GCCþ, especially in the early
stages of product manufacture, results in a final product with less
intensity of flavor, less color intensity and a more acidic taste. Thus,
sausages with high counts of GCCþ develop more volatile aroma
compounds, increasing the acceptability of the final product.
Sausages inoculated with probiotic LAB strains were more acid
than control sausages, and showed higher slice cohesiveness
(tactile texture descriptor) than control (P < 0.05). This could be
attributed to the pH values of the inoculated sausages which
approached to the isoelectric point of meat proteins faster (5.3),
providing protein coagulation that led to the cohesion of the sau-
sages. Once again, the high scores for the acidic taste of the inoc-
ulated treatments (mainly L. rhamnosus GG and L. plantarum 299V
 R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e56 55

Table 2
Sensory evaluation of fermented sausages at the end of ripening.

Attributes Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 RMSE* P

(Control) (GG ca. 105 CFU/g) (GG ca. 107 CFU/g) (299V ca. 105 CFU/g) (299V ca. 107 CFU/g)

Appearance
Cohesiveness 7.9b 8.0ab 8.2a 8.2a 8.1ab 0.4831 0.0099
Red color intensity 7.3a 5.7b 5.6b 7.0a 5.7b 0.9255 <0.0001
Brightness 6.2a 4.7b 4.6b 5.9a 4.7b 0.9679 <0.0001
Odor
Intensity 6.9a 5.3c 5.4c 6.3b 5.5c 0.9638 <0.0001
Cooked odor 0.3b 2.3a 2.2a 0.7b 2.1a 1.1998 <0.0001
Ripened odor 6.2a 3.5c 3.1d 5.0b 3.6c 1.0992 <0.0001
Tactile texture
Slice cohesiveness 6.9b 7.5a 7.7a 7.4a 7.4a 0.7016 <0.0001
Taste/flavor
Saltiness 3.9 4.0 4.0 4.0 3.8 0.8056 0.4657
Acid taste 2.4d 5.2b 6.5a 3.5c 5.3b 0.9974 <0.0001
Piquantness (pepper) 4.3a 4.0ab 3.9ab 4.3a 3.7b 0.9747 0.0316
Ripened 6.5a 3.7c 3.1d 5.7b 3.6cd 0.9484 <0.0001
Oral texture
Hardness 4.2b 5.1a 5.4a 4.9a 5.1a 0.9911 <0.0001
Elasticity 3.2c 4.2ab 4.4a 3.7bc 4.3ab 1.0314 <0.0001
Crumbliness 5.9a 5.1b 5.1b 5.5ab 5.1b 0.7714 <0.0001
Fibrousness 2.1c 2.8ab 3.3a 2.4bc 2.9ab 0.8198 <0.0001
Overall sensory quality 7.3a 4.7c 4.1d 6.8b 4.6cd 0.8370 <0.0001
aec
Within a row, means with different superscript letters significantly differ. *RMSE ¼ Root Mean Standard Error.

at high level) are related to the low values of pH in the final
products. Previous studies from our group (Garriga et al., 1996)
reported that L. plantarum CTC305, a meat isolate added at 105 CFU/
g as starter culture for the manufacture of Spanish-type fermented
sausage, as those of the present study, resulted in a final product
with an excessive acid taste, which is not well accepted by con-
sumers. On the contrary, in a study carried on North European
sausages inoculated with L. rhamnosus GG (at 107 CFU/g), with a
final pH below 5, the flavor was considered equal to that of control
sausages inoculated with a commercial non-probiotic starter cul-
ture (Erkkilä, Suihko et al., 2001). According to Askegaard and
Madsen (1998), Europe cannot be regarded as a homogeneous
sensory culture since important differences exist in consumption
patterns, behavior and attitudes, not only between countries but
also between regions within the same country. In our study, T5
(L. plantarum 299V dominating) showed lower piquantness than
the control samples (P < 0.05). The control samples and those
where L. plantarum 299V co-dominated with the endogenous
microbiota (T4) were scored with higher values for ripened flavor,
consistent with the sausages which had a longer ripening time and
higher final counts of GCCþ. Regarding the texture descriptors,
sausages inoculated with probiotic LAB cultures showed higher
values for hardness compared to control samples (P < 0.05). Elas-
ticity and fibrousness were higher in L. rhamnosus GG samples (T2,
T3) and L. plantarum 299V dominating (T5) than in control which
could be due to the lower pH values and slightly higher aw values at
the end of ripening. Crumbliness was significantly higher in control
samples (P < 0.05) compared to these inoculated with the two
levels of L. rhamnosus GG and L. plantarum 299V inoculated at high
level, but not significantly different from the treatment inoculated
with the low level of L. plantarum 299V (P > 0.05), which co-
dominated with the endogenous LAB.
The differences observed in the Quantitative Descriptive Sen-
sory Analysis influenced the overall sensory quality among treat-
ments and significant differences (P < 0.05) were observed
between the control samples and those inoculated with probiotic
LAB strains. The higher overall sensory quality was recorded by the
control sausages followed closely by treatment 4, where
L. plantarum 299V co-dominated with the endogenous microbiota.
L. plantarum 299V inoculated at low level (T4) showed physico-
chemical and sensory characteristics, which were in fact similar
to those recorded in the control treatment and improved the hy-
gienic quality of the sausages.
4. Conclusions
Fermented sausages inoculated with L. plantarum 299V at low
concentration (105 CFU/g) may be considered to be functional prod-
ucts, given the counts of the strain at the end of processing and during
shelf life (108 CFU/g) and the high overall sensory quality of the final
product, thus adding further value to this type of meat product as a
probiotic vehicle. However, human clinical studies are needed to
assess the health promoting effects of probiotic dry sausage.
Acknowledgments
The authors gratefully acknowledge the European Community
financial participation under the Sixth Framework Program for
Research, Technological Development and Demonstration Activ-
ities, for the Integrated Project Q-PORKCHAINS FOOD-CT-2007-
036245. The information in this document reflects only the view
of the authors and the Community is not liable for any use that may
be made of the information contained therein.
We would like to thank Sergi Raurich, Montse Badia and Quim
Arbonés for their technical assistance.
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