Innovative Food Science and Emerging Technologies 16 (2012) 89–95

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Innovative Food Science and Emerging Technologies

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The impact of fast drying (QDS process®) and high pressure on food safety ofNaCl-free processed dry fermented sausages

Katharina Stollewerk a, Anna Jofré a,⁎, Josep Comaposada b, Jacint Arnau b, Margarita Garriga a

a Food Safety Programme, IRTA, Finca Camps i Armet, 17121 Monells, Spainb Food Technology Programme, IRTA, Finca Camps i Armet, 17121 Monells, Spain

⁎ Corresponding author. Tel.: +34 972630052; fax: +E-mail address: anna.jofre@irta.cat (A. Jofré).

1466-8564/$ – see front matter © 2012 Elsevier Ltd. Alldoi:10.1016/j.ifset.2012.04.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 March 2012Accepted 30 April 2012

Editor Proof Receive Date 28 May 2012

Keywords:L. monocytogenesSalmonellaFast ripeningChorizoPressurizationFood safety

In the present study the food safety impact of the QDS process® combined with a high pressure treatment at600 MPa was evaluated in NaCl-free processed acid (pH 4.8) and low-acid (pH 5.2) chorizo. A challenge testwas performed where the raw meat batter was spiked with low levels of Listeria monocytogenes and Salmonella(b100 CFU/g) and chorizos were manufactured following either a traditional drying or a QDS process®. Afterdrying, half of the sliced chorizo samples were pressurized (600MPa, 5 min, 13 °C) and stored under refrigera-tion for 91 days. QDS processing proved to be adequate for the production of safe NaCl-free processed dryfermented sausages. Regarding pathogenicmicroorganisms elimination, itwas as effective as traditional process-ing for acid chorizo and even safer for low-acid chorizo. The high pressure treatment assured absence of bothpathogens in all samples during the whole storage time. Sausage reformulation to meet NaCl-free processingrequirements modified the progress of pH and technological microbiota.Industrial relevance: The QDS process®was designed to reduce themanufacturing time of sliced dry-cured meatproducts. It allows a just in timeworkflow, requires less space and energy and facilitates the rapid elaboration ofnew products, implying fast adaptation to marketing promotions. Among them, dry fermented sausages withreduced sodium chloride content is currently one of the major subjects investigated on the meat sector. Reduc-tion of NaCl and the use of replacers, however, could negatively affect food safety and quality, whichmust there-fore be properly evaluated. To extend shelf-life and improve safety, especially in products which arereformulated, high pressure processing could be a useful technology. The development of safe NaCl-free sliceddry fermented sausages in a short period of time is relevant for industry to meet consumer demands for conve-nient and healthy ready-to-eat products.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

A cause and effect relationship has been established between highdietary sodium intakes and increased blood pressure, which can leadto serious cardiovascular diseases (EFSA, 2011b). Hence, research onNaCl-reduced foods has increased in the recent years and a raising num-ber of patents has been awarded (Arnau, Comaposada, Serra, Bernardo,& Lagares, 2011; Toldra & Barat, 2009). In processed meat products,NaCl, due to its multifunctional character, contributes to different prod-uct characteristics and a simple reduction of the salt concentrationwould have important consequences on both, product quality (Doyle& Glass, 2010) and safety (Messier, Smith, & Tittiger, 1989). For dry fer-mented sausages, therefore, various strategies have been proposed toreplace the NaCl content, for example by using other chloride salts, ofwhich KCl being the most important one (Desmond, 2006). Potassiumis described to have lowering blood pressure properties and increase

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urinary sodium excretion. The intake of potassium represents a riskfor people who are predisposed to retain potassium, e.g. due to renaldysfunction, but has not been associatedwith adverse effects in normal,healthy children and adults (EFSA, 2005); however, doses should notexceed 3.1–3.5 g/day (EFSA, 2005). In any case, KCl can only partiallysubstitute NaCl due to its bitter taste, up to a maximum of 40% (Askar,El-Samahy, & Tawfik, 1994; Gou, Guerrero, Gelabert, & Arnau, 1996).Nevertheless, a 50% reduction of the NaCl content in small calibre fer-mented sausages (ca. 40-mm diameter) with the combination of KCl(40%) and potassium lactate (10%) achieved acceptable sensory results(Guàrdia, Guerrero, Gelabert, Gou, & Arnau, 2006). Lactate was men-tioned a flavor enhancer (Verma & Banerjee, 2012) and exerts a favor-able antimicrobial effect that has been described from various meatproducts (Jofré, Garriga, & Aymerich, 2008; Mbandi & Shelef, 2002;Stekelenburg, 2003; Stekelenburg & Kant-Muermans, 2001); however,regarding food safety of dry-cured meat products where the NaCl con-tent has been reduced or substituted, there is a lack of studies focussingon pathogenic microorganisms behavior (Fulladosa, Sala, Gou, Garriga,& Arnau, 2012; Gelabert, Gou, Guerrero, & Arnau, 2003; Stollewerk,Jofré, Comaposada, Arnau, & Garriga, 2012).

90 K. Stollewerk et al. / Innovative Food Science and Emerging Technologies 16 (2012) 89–95

Pathogenic microorganisms, such as L. monocytogenes andSalmonella have been a concern in ready-to-eat (RTE) products(EFSA, 2011a). Although the number of registered outbreaks associat-ed with dry fermented sausages is relatively small (Moore, 2004),different studies indicated that both pathogens could survive indry fermented sausages and may not be completely eliminatedduring the production process (Glass & Doyle, 1989; Nightingale,Thippareddi, Phebus, Marsden, & Nutsch, 2006;Whiting, 1993). Now-adays, U.S. processors must validate their own process for the specificdestruction of relevant pathogens, among them L. monocytogenes andSalmonella. A mild heat treatment combined with the acidity of fer-mented sausagesmay be a useful approach to eliminate the pathogensfrom the product (Glass & Doyle, 1989); however, since thermal treat-ment conditions are rather specific for each sausage type, validationprocedures are needed for individual sausage classes in agreementwith current USDA guidelines (Barbuti & Parolari, 2002; Glass &Doyle, 1989; Nightingale et al., 2006). High pressure (HP) processing,a non-thermal pasteurization method, can be applied to sliced meatproducts (Garriga & Aymerich, 2009) to improve microbiologicalproduct safety and extend shelf-life. In relation to reduced salt prod-ucts, HP has been proposed to have great potential as a complementa-ry technology to enhance product shelf-life (Verma & Banerjee, 2012).

The QDS process® is a patented technology (Comaposada et al.,2004) to accelerate the drying/ripening phase of dry-cured meat prod-ucts by drying slices directly. Sausages are fermented to the desired pHand then frozen, sliced and dried in a continuous system based on theapplication of convective air. The immediacy in obtaining the finishedproduct offers great flexibility and facilitates the optimisation of thedevelopment of new RTE products, for example salt-free processed dryfermented sausages (Arnau et al., 2011). In thisway, consumer demandscan be attended on time while saving money and space (MetalquimiaS.A., Girona, Spain). A recently published study assessing the food safetyimpact of QDS processing on acid and low-acid chorizo confirmed equal-ity to traditional processing (Stollewerk, Jofré, Comaposada, Ferrini, &Garriga, 2011); however, up to the present date, no studies exist onthe effect of QDS drying onNaCl-free processed dry fermented sausages.With the aim to evaluate possible food safety implications of these twonew technologies, a challenge test was performed where the meatbatters of NaCl-free processed acid and low-acid chorizo were spikedwith low levels of L. monocytogenes and Salmonella. Sampleswere eitherQDS or traditionally dried and half of the samples were pressurized tostudy the effect of a HP treatment at 600MPa.

2. Material and methods

2.1. Chorizo manufacturing, inoculation and drying

The flow chart representing different manufacturings for NaCl-freeprocessed chorizo and time-scale are shown in Fig. 1. The first part of

Spikedmeatbatter

GDL Acidification to pH 4.8

GDL Acidification to pH 5.2

QDS process® : flow chart and time s

GDL Acidification to pH 4.8

GDL Acidification to pH 5.2

TRADI process: flow chart and time

ThermalTreatment

43 oC 110 min

ThermalTreatment

43 oC65 min

Freezing And Slicing

QDSDryin45 m

TRADIDrying18 days

Slicin

Day 0 Day 2

Day 0 Day 2 Day 3

Day

Fig. 1. Schematic representation

manufacturing was the same for QDS dried (QDS) and traditionallydried (TRADI) chorizos. For each NaCl-free processed acid (A) andlow-acid (LA) chorizo batch a total of 15 kg was prepared by mixingthe following ingredients (in g/kg): meat (23% fat), 903; potassium lac-tate (77.8%, Purasal® Hi Pure P Plus, Purac bioquímica, S.A. Montmeló,Spain), 45; dextrose, 20; lactose, 20; gluconodeltalactone (GDL), 20(A) and 12 (LA); paprika, 10; water, 10; KCl, 5.1; tetrapotassium pyro-phosphate, 2.25; garlic, 1.5; potassium nitrite, 0.15; potassium nitrate,0.15; coloring carmine (6%), 1.25 and ascorbic acid, 0.5. LyophilisedStaphylococcus xylosus (Lyocarni SXH-38, Sacco, Activa) was used asstarter culture for A chorizo and LA chorizo at levels of 107 CFU/g to im-prove flavor. Each meat batter was spiked with 15 CFU/g of Salmonellaand ca. 30 CFU/g of L. monocytogenes, levels in keeping with thosefound in naturally contaminated products (CRL/AFSSA, 2008; Hoz,Cambero, Cabeza, Herrero, & Ordóñez, 2008). The bacterial cocktailwas prepared from three strain mixtures of each pathogen (Table 1)stored at −80 °C. Thawed cultures of the strains were diluted in10 ml of water. After mixing for 2 min at 0 °C the meat batter wasstuffed into 75-mm diameter collagen casings (1.0 kg per sausage).Subsequently, acidification/maturation was performed at 22 °C and90–95% relative humidity (RH) for 48 h for A and LA chorizo. Due tothe application of different amounts of GDL, which was converted ingluconic acid from themoment of its application until its complete con-sumption, pH values of 4.9 in A and 5.2 in LA chorizo were achieved.Subsequently, both TRADI and QDS sausages were submitted to a ther-mal treatment in a vapor oven at 53 °C until their core temperaturereached 43 °C, which took 65 min for TRADI and 110 min for QDS sau-sages, as previously described in Stollewerk et al. (2011). Sausages sub-jected to the traditional ripening-drying process were kept in a dryingchamber for 18 days at 13–15 °C and 70–76% RH. After reaching afinal weight loss of 30% they were frozen and sliced. Sausages subjectedto the QDS process® were immediately frozen after the thermal treat-ment, sliced and dried to a final weight loss of 30.1±0.9%, which tookapproximately 45 min. Two independentmanufacturings of the follow-ing batches: A-QDS, LA-QDS, A-TRADI and LA-TRADI were performedon two different days.

2.2. Pressurization and storage of slices

Sliced TRADI and QDS chorizo were vacuum-packed (40 g/bag) inPET/PE pouches (Sacoliva S.L., Spain) and kept for 18 h at 4 °C. Half ofthe samples were submitted to a HP treatment to 600 MPa for 5 minat 13 °C (Wave 6000 from Hiperbaric, Burgos, Spain). The chambervolume was 120 L, the come-up time was 3.83±0.13 min and thepressure-release was immediate (b10 s). Storage was performed for21 days at 4 °C and then for 70 days at 8 °C, following the tempera-ture profile recommended by guidance documents (AFNOR, 2004;CRL/AFSSA, 2008) and according to current retail conditions for slicedfermented sausages.

A-TRADI HP-

A-TRADI HP+

LA-TRADI HP-

LA-TRADI HP+

cale

scale

gin

g

VacuumPackaging40 g/bag

VacuumPackaging40 g/bag

Refrigerated Storage

21 days at 4 oC 70 days at 8 oC

Day 21

3

HP

HP A-QDS HP-

A-QDS HP+

LA-QDS HP-

LA-QDS HP+

HP

HP

of the experimental design.

Table 1Description of the strains.

Species L. monocytogenes S. enterica

Referencea CTC1011 CTC1034 CECT 4031 CTC1003 CTC1022 GN-0006Serotype/serovar 1/2c 4b 1a London Derby TyphimuriumOrigin Meat product Meat product Rabbit Meat product Meat product Pork gut

a CTC and CECT strains belong to collections from IRTA and Spanish type culture collection, respectively. GN strain was kindly provided by Dr. Badiola (CReSA, Bellaterra, Spain).

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2.3. Microbiological analyses

Sampling was performed in duplicate during production (in themeat batter, after acidification, after thermal treatment and after dry-ing), after pressurization and after 14, 28, 56 and 91 days of refriger-ated storage for both pressurized and non-pressurized chorizos.

For microbiological analysis, 25 g of the sample was diluted 1/10in Brain Heart Infusion (BHI, BD Becton, Dickinson and Company, NJ,USA) and homogenized for 1 min in a Masticator Classic (IUL S.A.,Barcelona, Spain). Appropriate dilutions of the homogenate in 0.1%Bacto Peptone (Difco Laboratories, Detroit, MI, USA) with 0.85%NaCl were plated onto the following media: Chromogenic Listeriaagar (Oxoid Ltd., Basingstoke, UK) incubated for 48 h at 37 °C forL. monocytogenes; CHROMagar™ Salmonella plus (Sharlab, Barcelona,Spain) incubated for 48 h at 37 °C for Salmonella; de Man, Rogosa andSharpe agar (MRS, Merck, Darmstadt, Germany) incubated for48–72 h at 30 °C in anaerobiosis for lactic acid bacteria (LAB) andMannitol salt phenol-red agar (MSA, Merck) incubated 48–72 h at30 °C for Gram-positive catalase-positive cocci (GCC+).

When L. monocytogenes and Salmonella counts were below the detec-tion limit in 135-mm diameter plates (10 CFU/g) their presence in theenriched (48 h at 37 °C) homogenized samples was investigated bygrowth in selectivemedia followedby real timePCR: for L.monocytogenes,dots of 20 μl were seeded onto Chromogenic Listeria agar, incubated at37 °C for 48 h and presumptive positive colonies were confirmed byreal time PCR (Rodríguez-Lázaro, Jofré, Aymerich, Hugas, & Pla, 2004).For the detection of Salmonella 200 μl of enriched culturewere inoculatedin 10 ml of Rappaport Vasiliadis (Oxoid) and incubated at 41.5 °C for 48 h.The presence of Salmonella in turbid tubes was determined by streaking50 μl of culture in CHROMagar™ Salmonella plus followed by confirma-tion by real time PCR (Malorny et al., 2004).

2.4. Determination of pH and aw

pH was measured at selected times with a puncture electrode5232 (Crison Instruments S.A., Alella, Spain) and a portable pHmeterPH25 (Crison) and aw with an Aqualab 3TE device (Decagon Devices,Inc. Pullman, Washington, USA).

2.5. Statistical analysis

Statistical tests were performed with Statistica 7.0 software (Statsoft,Tulsa, UK) and significance level was set at pb0.05. For bacterial counts(N, in log CFU/g), pH and aw, two-way ANOVA was performed for acid

Table 2aw values of acid and low-acid NaCl-free processed chorizo, treated (HP+) or non-treated

Chorizo type Acid (A)

Drying process QDS TRADI

HP treatment 0 MPa 600 MPa 0 MPa

Days of storage t0/ after HP 0.905±0.004 0.901±0.008 0.916±0.01128 days 0.907±0.001 0.905±0.003 0.919±0.01191 days 0.908±0.006 0.898±0.011 0.910±0.011

Values correspond to mean±SD (n=4).

and low-acid samples, using “drying system” (QDS or TRADI) and “pres-sure treatment” (HP− or HP+) as fixed factors. For determining statisti-cal differences of each batch during the manufacturing (of bacterialcounts, pH and aw) or storage period (pH and aw), one-way ANOVAwas performed, using “production step” or “days of storage” as fixed fac-tor. To allow graphical representation and statistical data processing ofabsence of the pathogens in 25 g of product (=0 CFU/g) in logarithmicunits, counts were transformed to log (N+1).

3. Results and discussion

3.1. Physicochemical parameters

aw at the end of drying was similar (p>0.05) in QDS and tradition-ally dried A and LA chorizo and no significant changes in aw valueswere recorded between different chorizo types (A and LA) and dryings(TRADI and QDS) during the storage period of 91 days under refrigera-tion (Table 2). pH values of A and LA chorizo started decreasing imme-diately due to GDL addition and slightly increased during drying,independently from the process (ca. 0.18 units, pb0.05, Table 3). Duringstorage, the pH continuously decreased in traditionally dried A and LAchorizo (pb0.05), while it did not significantly change in A- and LA-QDS processed samples. Consequently, after 91 days, pH levels werehigher in QDS than in TRADI chorizos (pb0.05, Table 3).

The achievement of low aw and pH during manufacturing of dry fer-mented meat products is important for the microbiological stability ofthe final product (Barbuti & Parolari, 2002). Therefore, both parametersmust be controlled, especially when hurdles for the growth of pathogenshave been modified. Regarding aw, in the present study QDS drying de-creased aw to the same level as the traditional drying method, but in asignificantly shorter time (45 min vs. 18 days). This fast and controlledaw decrease has been reported before to be compatible with theproduction of safe and wholesome dry-cured meat products (Ferrini,Comaposada, Arnau, & Gou, 2011; Stollewerk et al., 2011). pH evolutionshowed some differences when compared to standard chorizo(Stollewerk et al., 2011) or other dry fermented sausages manufacturedwith NaCl (Leistner, 1995), which is normal in products varying in com-position andmanufacturing (Lücke, 1998). The application of a HP treat-ment (600 MPa, 13 °C, 5 min) had no immediate or long-term effect(p>0.05) on the pH and aw of any of the samples (Tables 2 and 3).Physicochemical results from pressurized batches were in line withresults from other HP-treated fermented products (Jofré, Aymerich,Grèbol, & Garriga, 2009; Marcos, Aymerich, & Garriga, 2005; Stollewerket al., 2011).

(HP−) by high pressure at 600 MPa, during storage.

Low-acid (LA)

QDS TRADI

600 MPa 0 MPa 600 MPa 0 MPa 600 MPa

0.915±0.014 0.908±0.017 0.915±0.012 0.908±0.018 0.913±0.0080.918±0.010 0.912±0.015 0.912±0.021 0.915±0.010 0.911±0.0110.911±0.009 0.910±0.017 0.912±0.019 0.908±0.016 0.907±0.013

Table 3pH values of acid and low-acid NaCl-free processed chorizo, treated (HP+) or non-treated (HP−) by high pressure at 600 MPa, during storage.

Chorizo type Acid (A) Low-acid (LA)

Drying process QDS TRADI QDS TRADI

HP treatment 0 MPa 600 MPa 0 MPa 600 MPa 0 MPa 600 MPa 0 MPa 600 MPa

Production After acidif. 4.84±0.08Aa 4.84±0.08Aa 5.14±0.04Aa 5.14±0.04Aa

Step After therm.treatm. 4.93±0.10Aa 4.90±0.12Aab 5.23±0.05Ab 5.21±0.06Aa

Days of storage t0/ after HP 5.10±0.10Ab 5.09±0.10Ab 5.14±0.06Ad 5.10±0.11Ad 5.35±0.07Ac 5.33±0.07Ac 5.40±0.14Ab 5.35±0.15Ab

28 days 5.13±0.08Bb 5.39±0.11Cc 5.00±0.08Ac 5.02±0.06Acd 5.40±0.05Bc 5.49±0.06Bd 5.20±0.17Aa 5.23±0.13Aab

91 days 5.06±0.03BCb 5.10±0.10Cb 4.98±0.05Abc 5.00±0.06ABbc 5.33±0.04Bc 5.32±0.03Bc 5.17±0.13Aa 5.20±0.11Aa

Values correspond to mean±SD (n=4). For acid and low-acid chorizo, significant differences in rows are indicated by different capital letters and significant differences in columnsare indicated by different small letters (pb0.05).

92 K. Stollewerk et al. / Innovative Food Science and Emerging Technologies 16 (2012) 89–95

3.2. Evolution of technological microbiota

Initial GCC+counts were recorded at 7 log CFU/g in all chorizo sam-ples (corresponding to applied starter culture level) and were 2.5–2.9

MA

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A QDS LA QDS

0

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/g

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/g

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B

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0

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FU

/g

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B

Fig. 2. Behavior of technological microbiota during the manufacturing process (meat battering) and during refrigerated storage of non-pressurized (HP−) and pressurized (HP+) NaC

log lower in QDS and traditionally dried chorizo at the end of drying(pb0.05, Fig. 2). During subsequent storage, GCC+counts kept de-creasing in A-QDS and were after 91 days 2 log lower (pb0.05) thanin all other kinds of chorizo, where similar levels (ca. 4 log CFU/g)

0

2

4

6

8

Lo

g C

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/g

GC

C+

LA TRADI A TRADI

0

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2

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0 20 40 60 80

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Days of storage

, after acidification/maturation, after thermal treatment, after QDS and traditional dry-l-free processed chorizo.

Table 4Counts of L. monocytogenes (in log N+1 CFU/g) in acid and low-acid NaCl-free processed chorizo treated (HP+) or non-treated (HP−) by high pressure at 600 MPa.

Chorizo type: Acid Low-acid

Drying process: QDS TRADI QDS TRADI

Production step Before acidification 1.08±0.56Aa 1.08±0.56Aa 1.65±0.13Aa 1.65±0.13Aa

After acidification 0.26±0.52Ab 0.26±0.52Ab 1.69±0.34Ba 1.69±0.34Ba

After thermal treatment ND ND 1PRE/3ND* 0.52±0.60b

After drying ND ND ND ND

HP treatment: 0 MPa 600 MPa 0 MPa 600 MPa 0 MPa 600 MPa 0 MPa 600 MPa

Storage t0/after HP ND ND ND ND ND ND ND ND14 days ND ND ND ND ND ND 1PRE/3ND* ND28 days ND ND ND ND ND ND ND ND56 days ND ND ND ND ND ND ND ND91 days ND ND ND ND ND ND ND ND

Values correspond to mean±SD (n=4). For acid and low-acid chorizo, significant differences in rows are indicated by different capital letters and significant differences in columnsare indicated by different small letters (pb0.05). ND: not detected in 25 g; PRE: presence in 25 g.*L. monocytogenes was only detected in 1 out of 4 replicates.

93K. Stollewerk et al. / Innovative Food Science and Emerging Technologies 16 (2012) 89–95

maintained until the end of storage. LAB counts at the beginning of theexperiment were low (3 log CFU/g, Fig. 2). Acidification and thermaltreatment produced a significant decrease of LAB levels in QDS samples(Fig. 2). During drying, LAB counts increased in LA-TRADI chorizo to4.37 log CFU/g while maintained at low levels (ca. 2 log CFU/g) in A-TRADI and in both, A- and LA-QDS. During the following storage period,LAB kept the values, hence, higher counts (pb0.05) were observed inLA-TRADI chorizo, the batch where the highest pH decrease wasrecorded during storage (0.23 units, Table 3).

Differences in the production process in combination with theNaCl-free fermented sausage composition determined the behaviorof technological microbiota and allowed the growth of endogenousLAB in LA-TRADI chorizo during drying. Lactate has been shown to in-hibit LAB (Shelef, 1994; Taormina, 2010); however, this effect has onlybeen described in NaCl-containing products. In a study on chicken dryfermented sausages elaborated with 18 g/kg of sodium lactate, only areduction in the growth of inoculated Lactobacillus sakei (104 CFU/g)during the drying period (18 days at 14 °C) was observed (Deumier& Collignan, 2003). Similarly, in cooked ham, growth of inoculatedLactobacillus curvatus (104 CFU/g) containing 33 g/kg of sodium lac-tate was significantly lower than in the product without sodium lac-tate, but reached ca. 7.4 log CFU/g after 42 days of storage at 4 °C(Stekelenburg & Kant-Muermans, 2001). In vacuum-packed beef andin combination with GDL, lactate showed an enhanced inhibitory ef-fect on LAB (Garcia Zepeda et al., 1994). Consequently in the evaluatedchorizos, containing GDL and lactate, growth of LAB during traditionaldrying of NaCl-free LA products but not in any other chorizo typecould be due to the combination of higher pH, shorter thermal treat-ment, higher aw during processing and longer drying period. These

Table 5Counts of S. enterica (in log N+1 CFU/g) in acid and low-acid NaCl-free processed chorizo

Chorizo type Acid

Drying process: QDS

Production step Before acidification 1.04±0.53Aa

After acidification 0.97±0.49Aa

After thermal treatment NDAfter drying ND

HP treatment 0 MPa 600 MPa 0 MPa

Storage t0/after HP ND ND ND14 days ND ND ND28 days ND ND ND56 days ND ND ND91 days ND ND ND

Values correspond to mean±SD (n=4). For acid and low-acid chorizo, significant differenceare indicated by different small letters (pb0.05). ND: not detected in 25 g; PRE: presence in*Salmonella was only detected in 1 out of 4 replicates.

microbiological hurdles not only affected LAB but also GCC+, ofwhich counts only decreased in A-QDS, the chorizo submitted to theharshest conditions including lower pH and longer thermal treatment.

A HP treatment at 600 MPa only had an immediately reducing ef-fect on LAB from LA-TRADI chorizos (1.67 log, Fig. 2), the only batchwhere microorganisms grew during the drying period of 18 days.Subsequently LAB counts continued decreasing and from day 14,levels were no longer statistically different from the rest of the slicedchorizo samples, independent of type (A and LA) and drying method(TRADI and QDS). A similar reduction was observed in standard cho-rizo and NaCl-free processed dry-cured ham (Stollewerk et al., 2011;Stollewerk et al., 2012). On GCC+, HP had only a slight immediate ef-fect (p>0.05); however, during storage, lower GCC+levels werefound in QDS than in TRADI processed and in A than in LA chorizo(pb0.05 at day 91, Fig. 2), which confirms an enhancing effect of HPby other preservative factors used, i.e. thermal treatment or pH reduc-tion (Farkas & Hoover, 2000).

3.3. Fate of food-borne pathogens

To simulate a natural contamination of rawmaterial, themeat batterswere spiked with low levels of L. monocytogenes and Salmonella (b2 logCFU/g). During the acidification/maturation phase, L. monocytogeneswas reduced in A chorizo (0.82 log, pb0.05) and could no longer bedetected (in 25 g of product) in A-QDS and A-TRADI batches after ther-mal treatments (Table 4). In LA chorizo, in contrast, the pathogen wasnot reduced after acidification, but was eliminated after both QDS andTRADI drying processes. Salmonella did not significantly decrease duringacidification (Table 5) but was eliminated after thermal treatments

treated (HP+) or non-treated (HP−) by high pressure at 600 MPa.

Low-acid

TRADI QDS TRADI

1.04±0.53Aa 1.04±0.53Aa 1.04±0.53Aa

0.97±0.49Aa 0.86±0.37Aa 0.86±0.37Aab

1PRE/3ND* ND 0.30±0.00b

ND ND 1PRE/3ND*

600 MPa 0 MPa 600 MPa 0 MPa 600 MPa

ND ND ND 1PRE/3ND* NDND ND ND ND NDND ND ND 1PRE/3ND* NDND ND ND ND NDND ND ND ND ND

s in rows are indicated by different capital letters and significant differences in columns25 g.

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from A-QDS and LA-QDS and reduced in A-TRADI (presence in 25 g ofproduct detected in one replicate) and LA-TRADI (0.56 log) chorizo. Sal-monella has been described to have a lower growth limit in broth(ICMSF, 1996) and higher survival in acid cider than L. monocytogenes(Roering et al., 1999). However, acid tolerance is highly dependent onthe food matrix and in nutrient broth it has been shown to decreasewith increasing temperature (Gabriel & Nakano, 2010). After the tradi-tional drying process of 18 days the pathogen was absent (from 25 g ofproduct) in all A chorizos, while still detected in one out of four repli-cates in LA-TRADI. During the following refrigerated storage periodL. monocytogenes was detected at day 14 and Salmonella at day 28 inLA-TRADI chorizo slices.

For standard chorizo, QDS processing was recently demonstratedto be as safe as traditional processing (Stollewerk et al., 2011); how-ever, the product reformulation to meet NaCl-free requirements inthe current study could have affected pathogenic microorganisms be-havior. In general, food safety of dry fermented sausages depends onthe combined effect of various hurdles of which low aw and low pHplay the most important role. In the present study, pH and aw didnot differ during storage of QDS and traditionally processed chorizoslices and remained at levels which theoretically do not allowL. monocytogenes and Salmonella growth (ICMSF, 1996); however, al-though aw was lower in the present NaCl-free processed slices than inpreviously evaluated standard chorizo (Stollewerk et al., 2011), bettersurvival of pathogens, especially in LA-TRADI was observed. Taking intoconsideration (i) the adaptation of the chorizo formula to NaCl-free pro-cessing i.e. the exclusion of NaCl and LAB starter cultures and addition oflactate, KCl and GDL, (ii) the important role of food matrix compositionon microorganism behavior (Brocklehurst, 2004) and (iii) the ability ofL. monocytogenes and Salmonella to survive under low aw conditions(ICMSF, 1996; Rainaldi, Luciani, & Picconi, 1991), the accumulated effectof these factors could jeopardize the food safety of QDS and TRADI A andLA chorizos. According to Martin (2001), salt addition while processingmeat is essential for achieving microbiological stability (through de-creasing aw), solubilizing muscle proteins which in turn allows gel for-mation and development of an optimum texture and imparting a saltytaste in the final product. LAB, apart from their important organolepticcontributions, represent a significant competitive microbiota for unde-sired or hazardous acid-sensitive bacteria (Lücke, 1998); however, al-though NaCl was substituted and low levels of LAB (ca. 2 log CFU/g)were recorded, L. monocytogenes and Salmonellawere eliminated duringmanufacturing and stayed absent during the whole storage of both QDSand TRADI A chorizos.

The pH value of the chorizo influenced L. monocytogenes and Salmo-nella survival. From a food safety point of view, this observation high-lights the importance of the acidity hurdle in dry fermented sausagesand the requirement of additional preservative factors (thermal treat-ment, HP) for low-acid products. In the present study, theQDSprocess®was safer than the TRADI process to produce low-acid NaCl-freeprocessed chorizo, where Salmonella survived manufacturing and bothpathogens were sporadically detected at the first weeks of storage.This observation was attributed to the harsher conditions associatedwith the QDS process® (more intense thermal treatment and moretime at aw≤0.9). Inactivation studies on L. monocytogenes in beakersausage likewise showed that the application of a 57.2 °C (for 4 h) ther-mal treatment after fermentation produced the same reduction (>2 logCFU/g) as a lower and longer thermal treatment (51.7 °C for 8 h, Glass &Doyle, 1989). In the case of Salmonella, >7 log reductionswere achievedin Lebanon Bologna, when fermentation was followed by final heatingtreatments at 43.3 °C for 20 h or at 48.9 °C for 3 h (Ellajosyula et al.,1998). Other environmental factors like acidity influence the heatsusceptibility of pathogenic microorganisms (Gabriel & Nakano,2010), which explains the higher inactivation of L. monocytogenesand Salmonella in A than in LA chorizo after the thermal treatment.

In literature, strategies to reduce the NaCl content in dry fer-mented sausages have been reported, most by using other chloride

salts (Verma & Banerjee, 2012); however, this is the first time thatfood safety of dry fermented sausages manufactured without theuse of NaCl is evaluated. The appropriate replacement of the NaCl an-timicrobial properties could at least be partially debited to the pres-ence of lactate. In dry-cured meat products, the effect of lactateagainst L. monocytogenes and Salmonella has been studied in dry fer-mented sausages (Deumier & Collignan, 2003) and in NaCl-freeprocessed dry-cured ham (Stollewerk et al., 2012). The fact that lac-tate has been demonstrated to be more effective in combinationwith other additives such as NaCl (Tan & Shelef, 2002), nitrite(Sofos, 1983) and GDL (Garcia Zepeda et al., 1994; Juncher et al.,2000), raises the question that it possibly exerts a similar enhancedeffect when combined with KCl.

Taking into consideration the microbiological criteria EC 2073/2005 demanding b100 CFU/g of L. monocytogenes at the moment ofconsumption for RTE products which cannot support its growth andabsence of Salmonella in 25 g of product, in the present study, all cho-rizo types except LA-TRADI would comply with this regulation. Toeliminate pathogenic microorganisms from LA-TRADI, an HP treat-ment of 600 MPa would be required. In meat products, the bactericid-al effect of HP has been related to the ripening stage at which it isapplied and to the intrinsic properties of the food matrix (Garriga &Aymerich, 2009). Acidity intensifies the HP effect while low aw wasreported to exert a protective effect on microorganisms (Farkas &Hoover, 2000; Hereu, Bover-Cid, Garriga, & Aymerich, 2012). Addition-ally, it has been reported that certain ingredients, such as lactate, canprotect pathogens against HP, in dry-cured ham (Stollewerk et al.,2012) and in cooked ham (Jofré et al., 2008), which should be takeninto account in reformulated meat products, where potassium lactateis used as a NaCl replacer. In the present study, however, the applicationof a HP treatment at 600 MPa achieved absence of L. monocytogenes andSalmonella in low-acid vacuum-packed slices of traditionally dried cho-rizo and demonstrated to be useful for reformulated products to complywith food safety standards.

Many factors work in concert to inhibit or prevent bacterialgrowth and the production of safe foods requires ascertaining theright balance among them. As demonstrated in the present study,food reformulation can have an important impact on physicochemicalparameters, technological and pathogenic microbiota; however, froma food safety point of view, under low contamination levels, themanufacturing of safe NaCl-free processed acid and low-acid chorizoslices with the QDS process® has been shown to be possible.

Acknowledgments

This work was supported by CDTI (Centro para el DesarrolloTecnológico Industrial), Ingenio 2010 programme, reference CENIT2007–2016 (Futural) from the Spanish Ministry of Industry, Tourism& Trade. K. Stollewerk is recipient of a doctoral fellowship awardedby the INIA. The authors thank G. Ferrini for the support in experi-mental design and Y. Beltran for the technical assistance.

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