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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 Lactobacillusrhamnosus 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, Spainb Food Technology Program, Finca Camps i Armet, E-17121 Monells, Girona, Spain

a r t i c l e i n f o

Article history:Received 20 December 2012Received in revised form5 March 2013Accepted 7 May 2013

Keywords:Meat fermentationLactobacilliCompetitivenessRAPD-PCRSensory analysis

* Corresponding author. Tel.: þ34 972630052; fax:E-mail address: margarita.garriga@irta.cat (M. Gar

0023-6438/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2013.05.014

a b s t r a c t

Probiotic food products are a fast growing area. Although probiotic strains are currently used in dairyproducts, their commercial application in fermented meat products is not yet common. The aim of thisstudy was to assess the competitiveness of two probiotic Lactobacillus strains (Lactobacillus plantarum299V and Lactobacillus rhamnosus GG) during the manufacture of Spanish fermented sausages and theireffect on the hygienic and sensory qualities of the final products. The inoculated strains were successfullymonitored by Randomly Amplified Polymorphic DNA (RAPD)-PCR. Both strains prevented the growth ofEnterobacteriaceae throughout the entire ripening process. L. rhamnosus GG and L. plantarum 299V athigh inoculum (ca. 107 CFU/g) produced a sharp decrease of pH values and low growth of Gram-positiveCatalase-positive Cocci (GCCþ), leading to a negative effect on the sensory attributes evaluated. Never-theless, L. plantarum 299V inoculated at 105 CFU/g achieved and maintained high counts until the end ofripening and storage (ca.108 CFU/g), co-dominating (60%) with the endogenous microbiota, producingfunctional sausages with a satisfactory overall sensory quality. No major differences in physico-chemicalparameters 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.

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1. Introduction

During recent decades there has been a growing consumer de-mand for probiotic foods, a group of functional foods which arebelieved 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 productsafety is essentially gained by a fall in pH (4.5e5.0) and a decreaseof 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 processfavors the growth of the microorganisms which influence thesensory 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

þ34 972630373.riga).

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mainly involve the participation of Lactic Acid Bacteria (LAB),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 dealingwith the use of probiotics in fermented meat products to improvetheir nutritional value as functional foods have been reported (DeVuyst, 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 toact as a protection, improving the survival of probiotic lactobacillithrough the gastrointestinal tract (Klingberg & Budde, 2006). Asfermentedmeat products are processedwithout heating, they couldbe suitable products for assessing probiotic LAB as starter cultures(Ammor & Mayo, 2007). Counts greater than 107 CFU/g of LAB areusually 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

R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e5652

species such as Lactobacillus sakei, Lactobacillus curvatus, Lactoba-cillus plantarum and Lactobacillus casei are commonly used as startercultures in fermented sausages (Ammor & Mayo, 2007). Strains ofL. casei, Lactobacillus paracasei and Lactobacillus rhamnosus as po-tential functional starter cultures in meat products were suggestedbyMacedoet al. (2008) andRebucci et al. (2007). L. rhamnosusGG is awell 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, Petajäet al., 2001; Erkkilä, Suihko et al., 2001). In vitro studies carried outby Pennacchia et al. (2006) drew attention to the suitability ofseveral strains of the L. plantarum-group isolated from traditionaldry-fermented sausages, for their probiotic use as starter cultures inmeat products. L. plantarum 299V (ProViva) and L. rhamnosus GG(Gefilus, Vifit) are commercially sold as probiotics. However, in orderto choose a probiotic LAB for use as a starter culture in fermentedsausages, desirable technological, sensory and safety properties onthe final products are required. No reference about L. plantarum299V assayed as a putative probiotic starter culture in sausagemanufacturing has been found.

The aim of the present work was to assess the suitability of twoprobiotic LAB strains (L. plantarum 299V and L. rhamnosus GG) asstarter cultures for Spanish fermented sausages, by evaluating theircompetitiveness 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 and107 CFU/g). Each strain was grown overnight in MRS broth (Merck,Darmstadt, Germany) at 37 �C, harvested by centrifugation at5000 rpm for 10 min at 6 �C, washed twice in saline solution (0.85%NaCl), resuspended in saline solution and stored at�80 �Cwith 20%of glycerol until further use.

2.2. Sausage preparation

The fermented sausages were manufactured using 80% leanpork and 20% pork belly, ground in a meat grinder by passingthrough 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 of20 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 withL. plantarum 299V (inoculated at ca. 105 and ca. 107 CFU/g, respec-tively). After mixing, the meat batter was stuffed into 50 mmdiameter collagen casings (Colex 32, Fibran S. A., Sant Joan de lesAbadesses, Spain). The sausages were dipped into a solution ofPenicillium candidum spores (Danisco, France), fermented at 20e22 �C and 90e95% RH (until pH value ca. 5.0) and ripened at 12 �Cand 75e80% RH (until aw value ca. 0.90e0.93). Ripened productswere 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, BarcelonaSpain) 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 pHmeter 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 samplesduring the whole process. For each sampling time (B, EF, ER), thecasings were aseptically removed and 15 g of fuet were crumbledand homogenized (1/10 dilution) with 0.1% Bacto Peptone (DifcoLaboratories, Detroit, MI, USA) with 0.85% NaCl (Merck) in aMasticator Classic (IUL S.A., Barcelona, Spain) for 60 s. Serial deci-mal dilutions were made and LAB were enumerated by plating onde Man, Rogosa and Sharpe (MRS) agar (Merck) at 37 �C for 72 h inanaerobiosis (Anaero-Gen, Oxoid); Gram-positive catalase-positivecocci (GCCþ) were determined by plating on Mannitol Salt Agar(MSA, Merck) at 30 �C for 48 h and Enterobacteriaceae by plating inViolet Red Bile Dextrose Agar (VRBD, Merck) with a double layer at30 �C for 24 h. Before sensory analysis Listeria monocytogenes andStaphylococcus aureus were determined on Chromogenic ListeriaAgar (CLA, Oxoid, Basingstoke, UK) incubated at 37 �C for 48 h andBaird-Parker agar base with egg yolk tellurite emulsion (BP, Oxoid)at 37 �C for 48 h, respectively. The presence/absence of Salmonellawas determined in 25 g according to ISO 6579 (2002). At the end ofstorage 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 MRSagar plates at each sampling time. For DNA extraction, isolatedcolonies were suspended in 200 ml of 6% Chelex�-100 chelating ionexchange resin (Bio-Rad, Hercules, CA, USA), heated at 100 �C for10 min, cooled on ice and centrifuged at 14,000 g for 10 min. Twomicroliter of the resuspended supernatant containing the DNAwasused as a PCR template. Two random primers (Roche MolecularBiochemicals, 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 andstained with 0.1 mg/ml ethidium bromide (Sigma, St. Louis, MO,USA). Three lines of 1 kb DNA ladder (Invitrogen) were used asmolecular weight and normalization gel standards for RAPD pro-files. The banding profiles were visualized under UV light anddigitalized by Gelprinter photodocumentation equipment (TDI,Barcelona, Spain).

2.6. Sensory analysis

Eight trained assessors (ASTM, 1981; ISO 8586-1, 1993; ISO8586-2, 1994) took part in the sensory analysis on 2.5 mm thickslices. The generation and selection of the descriptors was carriedout 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 lowintensity of the descriptor and 10 means very high intensity of thedescriptor (Amerine, Pangborn, & Roessler, 1965). Means of scoresgiven by the assessors for each sausage were recorded. Sensoryevaluation was undertaken in 5 sessions per product and a com-plete block designwas used (Steel & Torrie,1983), where each tasterassessed all the treatments in each session. Samples were codedwith three-digit random numbers and were presented to the as-sessors balancing the first order and the carry over effects accordingto 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, andmicrobiological data recorded during sampling times includedtreatment, time and their interaction as fixed effects. For sensorydata, the model included the treatment and the taste session asfixed effects. Mean differences among treatments were assessed bythe post-hoc Tukey test (P < 0.05). The percentage of implantationof a given inoculated strainwas ascertained according to a samplingplan based on the binomial distribution (Peña Sánchez de Rivera,1986). The implantation breakpoint, defined as a percentage ofcolonies that showed the same RAPD profile as the added probioticcultures, 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 batterwas 6.07. At day 6 (end of fermentation), a significant decrease ofpH was observed in all the treatments (P < 0.05), the pH valuesranging from 4.6 to 5.2.

At the end of fermentation the treatments inoculated withL. rhamnosus GG (T2 and T3) and with the high level of L. plantarum299V (T5) showed the highest decrease of pH (ca. 4.6) withsignificantly lower values (P < 0.05) than the pH recorded for thelot which has been inoculated with the low level of L. plantarum299V (T4) (5.01) and the control one (5.20). Therefore, L. rhamnosusGG showed higher acidifying ability than L. plantarum 299V. The pHdecrease contributes to the inhibition of undesirable microorgan-isms, accelerates the reduction of nitrite to nitric oxide, affects theflavor of the product and facilitates meat binding capacity,improving firmness and sliceability (Lücke, 1998; Varnam &Sutherland, 1995) and thus contributes to the safety and qualityof the sausages.

During ripening, pH was significantly different among thetreatments (P< 0.05). At the end of the process, in treatments 1 and

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

Manufacturestep

Time(days)

Treatment

1. Control 2. L. rhamnosca. 105 CFU/g

pH B 0 6.03 � 0.02Ca 6.07 � 0.02B

EF 6 5.19 � 0.11Ab 4.63 � 0.05B

ER 34 5.21 � 0.05Ab 4.85 � 0.03C

aw B 0 0.969 � 0.00a 0.969 � 0.00a

EF 6 0.965 � 0.00Ab 0.962 � 0.00B

ER 20 0.907 � 0.00Cc 0.928 � 0.00A

LAB B 0 2.59 � 0.16Db 5.89 � 0.15C

EF 6 8.31 � 0.01Aa 8.19 � 0.09A

ER 38 8.84 � 0.23Aa 8.19 � 0.10BGCCþ B 0 3.54 � 0.06c 3.53 � 0.02b

EF 6 7.14 � 0.18Ab 4.89 � 0.02B

ER 23 7.63 � 0.14Ab 4.81 � 0.12C

Enterobacteriaceae B 0 2.50 � 0.08Bc 2.84 � 0.16A

EF 6 4.75 � 0.08Ab 2.63 � 0.22B

ER 20 6.43 � 0.19Ab 1.36 � 0.10C

Values are mean � SD of triplicates. Significant differences in rows are indicated by diffesmall letters (P < 0.05).Bacterial counts are expressed in log CFU/g, limit of detection 1.0 log CFU/g for EnterobaB ¼ stuffed batter, EF ¼ end of fermentation, ER ¼ end of ripening.

4 (control and L. plantarum 299V at low level) no changes wereobserved in pH values compared to the values reached at the end offermentation (day 6), whereas in treatments 2, 3 and 5, which werethe most acid after fermentation, pH values increased slightly,though remained at lower values than those of the spontaneouslyfermented sausages (control).

The aw of the sausages decreased (P < 0.05) from an initial valueof 0.97 to 0.91e0.93 (Table 1), taking different times to reach thesevalues depending on the treatment. Control treatment followed bytreatment 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-parisonwith the control, which could be related to the reduction ofthe water-binding capacity of proteins caused by a strongeracidification.

3.2. Bacterial growth during fermentation and ripening

Table 1 shows the results of microbial counts. Endogenous GCCþshowed a sharp increase (P < 0.05) from ca. 3 log CFU/g to7.14 log CFU/g at day 6 (EF) in control treatment (non inoculatedwith LAB). These levels were kept constant or significantly higher atthe 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 thetreatments inoculated with the low level of L. rhamnosus GG andL. plantarum 299V (treatments 2 and 4, respectively), without sig-nificant differences between them (P > 0.05). However, in thetreatments inoculated with the high level of probiotics, the countsremained constant compared to initial time, until the end offermentation (P > 0.05). At the end of ripening, the treatmentinoculated with the low inoculum of L. plantarum 299V presented aslight increase in the counts of 0.7 logs, showing significantlyhigher levels than the rest of the inoculated treatments(5.99 log CFU/g) (P < 0.05), whereas treatment 2 remained at thelevels achieved at the end of fermentation (ca. 4.8 log CFU/g).Treatments 3 and 5 (high levels of L. rhamnosusGG and L. plantarum299V, respectively) showed a significant increase (P < 0.05),reaching final counts of 4.92 and 4.32 log CFU/g, respectively. Thelow levels of GCCþ in treatments 2, 3 and 5 could be explained by

us GG 3. L. rhamnosus GGca. 107 CFU/g

4. L. plantarum 299Vca. 105 CFU/g

5. L. plantarum 299Vca. 107 CFU/g

Ca 6.05 � 0.02BCa 6.09 � 0.02ABa 6.13 � 0.02Aac 4.58 � 0.03BC 5.02 � 0.03Ab 4.65 � 0.05Bcb 4.69 � 0.01Db 5.01 � 0.03Bb 4.79 � 0.02Cb

0.966 � 0.00a 0.967 � 0.00a 0.967 � 0.00ab 0.951 � 0.00Cb 0.964 � 0.00ABa 0.964 � 0.00ABac 0.918 � 0.00Bc 0.908 � 0.00Cb 0.925 � 0.00ABbb 7.50 � 0.07A 5.82 � 0.04Cb 7.06 � 0.01Bba 7.48 � 0.41B 8.71 � 0.16Aa 8.51 � 0.02AaCa 7.88 � 0.09C 8.48 � 0.03Ba 8.29 � 0.11Ba

3.62 � 0.09b 3.63 � 0.24c 3.59 � 0.02ba 3.26 � 0.50Cb 5.29 � 0.03Bb 2.78 � 0.36Cba 4.92 � 0.05Ca 5.99 � 0.01Ba 4.32 � 0.20Daba 2.99 � 0.39Aa 2.52 � 0.23Ba 2.49 � 0.10Baa 1.09 � 0.19Db 2.71 � 0.01Ba 2.08 � 0.11Cbb <1.00Cb 1.83 � 0.26Bb <1.00Cc

rent capital letters and significant differences in columns are indicated by different

cteriaceae.

R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e5654

the strong acidification achieved during fermentation in compari-son with the control spontaneous fermented sausages (treatment1). At the end of storage and for all treatments the GCCþ countswere maintained at the levels achieved at the end of ripening.

As expected, the counts of LAB at the beginning of the processwere lower in the control treatment (endogenous microbiota) thanin those inoculated with probiotic cultures. Generally, at the end offermentation (day 6), the counts of LAB had increased significantly(P < 0.05), reaching counts of 108 CFU/g which were maintaineduntil the end of the ripening period. According to Raccach (1992), arapid 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, thecounts remained ca.108 CFU/g in all treatments, demonstrating thecompetitiveness of LAB under the conditions assayed. LAB countsand irrespective implantation of both strains were maintained tillthe end of storage at 1 �C at the levels achieved after ripening.

The competitiveness of the inoculated probiotic LAB strains wasspecifically monitored by RAPD-PCR profiling analysis (Aymerichet 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 asthe parental strain. At the end of ripening (ER), a 100% implantationwas 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 of8.48 log CFU/g, 59.6% (18 patterns) of the RAPD profiles wereidentified as L. plantarum 299V. Thus, the probiotic strain achievedlevels of 1.7� 108 CFU/g co-dominatingwith other endogenous LAB(40.4%). Although the minimum recommended daily dose of pro-biotic bacteria is not known, it is estimated to be 109e1010 viablecells 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 containing108 viable cells/g, such as those of the present work, the minimumdosage for probiotic use would be achieved by eating 10e25 g ofsausage per day, which is quite feasible and compatible with anutritionally balanced diet.

The RAPD-PCR studywas useful in confirming the implantation ofthe probiotic LAB strains in fermented sausages, verifying that thismethod is adequate to discriminate lactobacilli at strain level(Aymerich et al., 2006). The obtained results demonstrated that theassayed probiotic cultures grew rapidly in fermented sausages anddominated or co-dominated (L. plantarum 299V at low inoculumlevel)with the endogenous LABduring the fermentation and ripeningprocesses, proving their suitability as probiotic starter cultures. Otherstudieshaveensured the suitabilityofprobiotic strainsof L. rhamnosusand L. plantarum for use as probiotic cultures in fermented sausagesaccording to their adaptation to the sausage environment and theirfast growth rate and acidification. Erkkilä, Petajä et al. (2001) andErkkilä, Suihko et al. (2001) showed that L. rhamnosus GG,L. rhamnosus E-97800 and L. plantarum E-98098were suitable for useas starter cultures in more acidic North European dry sausages.Klingberg et al. (2005) identified L. plantarum strains originating fromthe dominant non-starter LAB fermentedmeat products as promisingcandidates 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 below103 CFU/g (Table 1), values considered hygienically correct for rawmeat. At the end of fermentation (day 6), in the control treatment,Enterobacteriaceae counts increased significantly (P < 0.05; to4.75 log CFU/g) and maintained levels significantly higher than inthe 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 ofripening (ER), Enterobacteriaceae counts were below 2 log CFU/g inall the inoculated treatments, significantly lower (P < 0.05)compared to the counts for the control lot (>6 log CFU/g). Theseresults confirmed the inhibitory effect of the inoculated probioticcultures 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 initialstages 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 growthwas observed in treatments with starter culture.

In thefinal products, the foodbornepathogens (L.monocytogenes,S. aureus) were not detected in any treatment (values below thedetection limit<1 log CFU/g). Salmonellawas not detected (absencein 25 g) in any final product, confirming the safety of fermentedsausages 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 sausagesat 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 berelated to the differences observed in pH and the lower levels ofGCCþ compared to the control treatment.

Regarding appearance, the control samples showed slightlylower scores (P < 0.05) of visual cohesiveness which was similar inall the inoculated lots. Control samples (treatment 1) and thoseinoculated with the low inoculum level of L. plantarum 299V(treatment 4), but co-dominating with the endogenous LAB at theend of ripening, presented a higher intensity of red color andbrightness than T2, T3 and T5 (P < 0.05). Concerning the odor de-scriptors, the highest values for overall odor intensity as well as forripened odor, were scored in control sausages and in those of T4(P < 0.05) with slight differences. The rest of the treatments hadvalues significantly lower (P < 0.05) for these descriptors andhigher values of cooked odor (P < 0.05) which could be due to thelower counts of GCCþ. These bacteria have catalase and nitratereductase activities which contribute to preventing oxidation infermented sausages. The lower counts of GCCþ, produced by thestrong 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 alower development of the characteristic red color and ripened odorin these sausages. According to Metaxopoulos, Samelis, & Papadelli(2001), the predominance of LAB on GCCþ, especially in the earlystages of product manufacture, results in a final product with lessintensity of flavor, less color intensity and a more acidic taste. Thus,sausages with high counts of GCCþ develop more volatile aromacompounds, increasing the acceptability of the final product.

Sausages inoculated with probiotic LAB strains were more acidthan control sausages, and showed higher slice cohesiveness(tactile texture descriptor) than control (P < 0.05). This could beattributed to the pH values of the inoculated sausages whichapproached 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

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

Attributes Treatment 1(Control)

Treatment 2(GG ca. 105 CFU/g)

Treatment 3(GG ca. 107 CFU/g)

Treatment 4(299V ca. 105 CFU/g)

Treatment 5(299V ca. 107 CFU/g)

RMSE* P

AppearanceCohesiveness 7.9b 8.0ab 8.2a 8.2a 8.1ab 0.4831 0.0099Red color intensity 7.3a 5.7b 5.6b 7.0a 5.7b 0.9255 <0.0001Brightness 6.2a 4.7b 4.6b 5.9a 4.7b 0.9679 <0.0001OdorIntensity 6.9a 5.3c 5.4c 6.3b 5.5c 0.9638 <0.0001Cooked odor 0.3b 2.3a 2.2a 0.7b 2.1a 1.1998 <0.0001Ripened odor 6.2a 3.5c 3.1d 5.0b 3.6c 1.0992 <0.0001Tactile textureSlice cohesiveness 6.9b 7.5a 7.7a 7.4a 7.4a 0.7016 <0.0001Taste/flavorSaltiness 3.9 4.0 4.0 4.0 3.8 0.8056 0.4657Acid taste 2.4d 5.2b 6.5a 3.5c 5.3b 0.9974 <0.0001Piquantness (pepper) 4.3a 4.0ab 3.9ab 4.3a 3.7b 0.9747 0.0316Ripened 6.5a 3.7c 3.1d 5.7b 3.6cd 0.9484 <0.0001Oral textureHardness 4.2b 5.1a 5.4a 4.9a 5.1a 0.9911 <0.0001Elasticity 3.2c 4.2ab 4.4a 3.7bc 4.3ab 1.0314 <0.0001Crumbliness 5.9a 5.1b 5.1b 5.5ab 5.1b 0.7714 <0.0001Fibrousness 2.1c 2.8ab 3.3a 2.4bc 2.9ab 0.8198 <0.0001Overall 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.

1 * denotes the key references: Ammor, M. S., & Mayo, B. (2007): this reviewdiscusses the criteria for the selection of LAB starter cultures for fermented sau-sages. Aymerich, T., Martín, B., Garriga, M., Vidal-Carou, M. C., Bover-Cid, S., & Hugas,M. (2006): this research paper proves RAPD-PCR as an efficient method todiscriminate lactobacilli at strain level. Erkkilä, S., Petajä, E., Eerola, S., Lilleberg, L.,Mattila-Sandholm, T., & Suihko, M. L. (2001): this research paper studies the flavourprofiles of dry sausages fermented by strains of L. rhamnosus and L. plantarum.Rouhi, M., Sohrabvandi, S., & Mortazavian, A. M. (2013): this review discusses theviability of probiotics in fermented sausages, the main factors affect their viabilityand the sensorial characteristics of the final product. Klingberg, T. D., Axelsson, L.,Naterstad, K., Elsser, D., & Budde, B. B. (2005): research paper that studies twostrains of L. plantarum, isolated from Norwegian fermented sausages, as starterculture for Scandinaviant-type sausages.

R. Rubio et al. / LWT - Food Science and Technology 54 (2013) 51e56 55

at high level) are related to the low values of pH in the finalproducts. 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 fermentedsausage, as those of the present study, resulted in a final productwith an excessive acid taste, which is not well accepted by con-sumers. On the contrary, in a study carried on North Europeansausages inoculated with L. rhamnosus GG (at 107 CFU/g), with afinal pH below 5, the flavor was considered equal to that of controlsausages inoculated with a commercial non-probiotic starter cul-ture (Erkkilä, Suihko et al., 2001). According to Askegaard andMadsen (1998), Europe cannot be regarded as a homogeneoussensory culture since important differences exist in consumptionpatterns, behavior and attitudes, not only between countries butalso between regions within the same country. In our study, T5(L. plantarum 299V dominating) showed lower piquantness thanthe control samples (P < 0.05). The control samples and thosewhere L. plantarum 299V co-dominated with the endogenousmicrobiota (T4) were scored with higher values for ripened flavor,consistent with the sausages which had a longer ripening time andhigher final counts of GCCþ. Regarding the texture descriptors,sausages inoculated with probiotic LAB cultures showed highervalues 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 whichcould be due to the lower pH values and slightly higher aw values atthe end of ripening. Crumbliness was significantly higher in controlsamples (P < 0.05) compared to these inoculated with the twolevels of L. rhamnosus GG and L. plantarum 299V inoculated at highlevel, but not significantly different from the treatment inoculatedwith 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 observedbetween the control samples and those inoculated with probioticLAB strains. The higher overall sensory quality was recorded by thecontrol sausages followed closely by treatment 4, whereL. 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 lowconcentration (105 CFU/g) may be considered to be functional prod-ucts, given the counts of the strain at the end of processing andduringshelf life (108 CFU/g) and the high overall sensory quality of the finalproduct, thus adding further value to this type of meat product as aprobiotic vehicle. However, human clinical studies are needed toassess the health promoting effects of probiotic dry sausage.

Acknowledgments

The authors gratefully acknowledge the European Communityfinancial participation under the Sixth Framework Program forResearch, Technological Development and Demonstration Activ-ities, for the Integrated Project Q-PORKCHAINS FOOD-CT-2007-036245. The information in this document reflects only the viewof the authors and the Community is not liable for any use that maybe made of the information contained therein.

We would like to thank Sergi Raurich, Montse Badia and QuimArbonés for their technical assistance.

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