Innovative Food Science and Emerging Technologies 16 (2012) 26–32

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Innovative Food Science and Emerging Technologies

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Combined effect of high pressure treatments and the lactoperoxidase system on theinactivation of Listeria monocytogenes in cold-smoked salmon

Raquel Montiel, Daniel Bravo, María de Alba, Pilar Gaya, Margarita Medina ⁎Departamento Tecnología de Alimentos, INIA, Carretera de La Coruña Km 7, Madrid, 28040 Spain

⁎ Corresponding author. Tel.: +34 913476774; fax: +E-mail address: mmedina@inia.es (M. Medina).

1466-8564/$ – see front matter © 2012 Elsevier Ltd. Alldoi:10.1016/j.ifset.2012.03.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 January 2012Accepted 31 March 2012

Editor Proof Receive Date 10 May 2012

Keywords:L. monocytogenesCold-smoked salmonHigh pressureLactoperoxidase system

The effect of high hydrostatic pressure (HHP) processing at 250 and 450 MPa for 10 min combined with thelactoperoxidase system (LPS) on the inactivation of Listeria monocytogenes H66a and the characteristics ofcold-smoked salmon during 35 d at 5 °C were investigated. A synergistic antimicrobial effect of 450 MPaand LPS against L. monocytogenes was registered, preventing the pathogen recovery. Biogenic amine forma-tion was avoided. Lightness (L*) values increased by HHP and LPS treatments applied individually or in com-bination, resulting in a brighter and less transparent appearance of smoked salmon. Redness (a*) andyellowness (b*) were also affected. Hardness and shear strength increased with HHP processing and LPStreatments. HHP at 450 MPa for 10 min in combination with the LPS added to smoked salmon might beused as a hurdle technology approach against L. monocytogenes, increasing the safety and the shelf-life duringrefrigerated storage.Industrial relevance: Antimicrobial activity against L. monocytogenes of high pressure treatments in combina-tion with a biopreservative, the lactoperoxidase system in refrigerated cold-smoked salmon is presented.Color and texture of cold-smoked salmon were affected, whereas the safety and shelf-life of the productwere improved.

© 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Listeriosis is an atypical foodborne disease that affects more fre-quently pregnant women, new-born infants, children and immuno-suppressed adults (EFSA, 2010) with a death rate close to 20–30%(Lianou & Sofos, 2007). Listeria monocytogenes is frequently detectedin cold-smoked salmon and can pose an important risk to human healthif the pathogen reaches high levels in the product (Lakshmanan &Dalgaard, 2004). The presence of L. monocytogenes in cold-smokedsalmon can be mainly ascribed to contamination during processing(Rorvik, 2000; Vogel, Huss, Ojeniyi, Ahrens, & Gram, 2001). Cut-ting, slicing and packaging increase the risks of smoked salmoncontamination.

The difficulty of controlling the pathogen in smoked salmon andother RTE (ready-to-eat) foods that are consumed without furthercooking has increased the interest in alternative preservation tech-nologies as high hydrostatic pressure (HHP) processing and biopre-servation to improve the safety and shelf-life of food products. HHPprocessing is applied to control post-processing contaminants inmany foods. The impact of HHP in the characteristics of seafood prod-ucts has been focused on its effect on muscle color (Amanatidou et al.,2000; Ritz, Jugiau, Federighi, Chapleau, & de Lamballerie, 2008),

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texture (Gómez-Estaca, Montero, Giménez, & Gomez-Guillén, 2007;Lakshmanan, Patterson, & Piggott, 2005), lipids (Sequeira-Muñoz,Chevalier, LeBail, Ramaswamy, & Simpson, 2006) and shelf-life(Amanatidou et al., 2000; Smelt, 1998). Studies on the inactivationof L. monocytogenes by HHP in cold-smoked salmon have demonstrat-ed that treatments at 250 MPa were not effective to inactivateL. monocytogenes but extended lag phases at refrigeration tempera-tures (Lakshmanan & Dalgaard, 2004). HHP (400–900 MPa) at veryshort times were effective to control Listeria innocua in smoked salmon(Gudbjornsdottir, Jonsson, Hafsteinsson, & Heinz, 2010).

The lactoperoxidase system (LPS), consisting of lactoperoxidase,thiocyanate (SCN−) and hydrogen peroxide (H2O2), is a natural anti-microbial system that occurs naturally in milk and other secretionssuch as saliva and tears. Lactoperoxidase catalyzes the oxidation ofSCN− by H2O2 into short-lived intermediate products with antibac-terial properties, such as hypothiocyanite (OSCN−) anion andhypothiocyanous (HOSCN) acid. These reactive products oxidize sul-phydryl groups (−SH) of proteins in the bacterial cell membraneand inhibit several important enzymes in cellular metabolism, inter-fering the transport of nutrients, the DNA and RNA synthesis andthe respiratory chain (Naidu, 2000). The use of LPS has been promot-ed as an effective method to extend the shelf-life of rawmilk in devel-oping countries (FAO/WHO, 1991). Its bactericidal activity againstL. monocytogenes in milk and dairy products has been widely reported(Arqués, Rodríguez, Nuñez, & Medina, 2008; García-Graells, VanOpstal, Vanmuysen, & Michiels, 2003; Zapico, Medina, Gaya, &

27R. Montiel et al. / Innovative Food Science and Emerging Technologies 16 (2012) 26–32

Nuñez, 1998), and is related to the initial inoculum, the culture medi-um and the storage temperature (Seifu, Buys, & Donkin, 2005). Theuse of LPS has been also approved as processing aid for meat andmeat products by the Food Standards Australia New Zealand(FSANZ, 2002). Although this antimicrobial system has been pro-posed in fishery and meat products to inhibit the growth of the path-ogen, only few studies have been published. The LPS added to freshmeat exhibited a bacteriostatic effect on L. monocytogenes (Elliot,McLay, Kennedy, & Simmonds, 2004; Kennedy, O'Rourke, McLay, &Simmonds, 2000). In smoked salmon, counts of L. monocytogeneswere also reduced during refrigeration by whey protein films incor-porating the LPS (Min, Harris, & Krochta, 2005).

Combined preservative factors (hurdles) may provide greater pro-tection than individual treatments, contributing to improve the safetyand quality of foods (Leistner, 2000). The strong cooperative effect ofHHP and the LPS on foodborne bacteria inactivation was demonstratedby García-Graells et al. (2003), being more effective than the combina-tion of pressurization with lysozyme, nisin and other bacteriocins.

The aim of this work was to investigate the effect of combinedHHP and the LPS on the survival of L. monocytogenes in cold-smokedsalmon during 35 d of chilled storage. The changes in total viablecounts and the characteristics of smoked salmon (pH, water activity,biogenic amines, color and texture) were also investigated.

2. Materials and methods

2.1. Microorganisms

L. monocytogenes INIA H66a (from the INIA Culture Collection,Instituto Nacional de Investigación y Tecnología Agraria y Alimen-taria, Madrid, Spain) was used as test organism in this study. Thestrain was maintained as stock culture at −80 °C in Trypticase SoyYeast Extract Broth (TSYEB, Biolife s.r.l., Milano, Italy) supplementedwith 30% glycerol. The bacterial strain was grown on TSYEB at 37 °Cfor 18 h before use in experiments.

2.2. Preparation of smoked salmon samples

Sliced cold-smoked salmon (Salmo salar, L.) was purchased from alocal supermarket in Madrid (Spain). Slices were aseptically cut into20-g pieces for microbiological analysis and 80-g pieces for pH,water activity, color, texture and biogenic amine content determina-tion. The 20-g samples were inoculated by adding 100 μl of the teststrain on the surface of smoked salmon in order to attain a final pop-ulation of approximately 107 cfu/g.

2.3. HHP processing

The smoked salmon samples were vacuum-packed in BB325 bags(Cryovac Sealed Air Corporation, Milan, Italy) and pressurized at 250and 450 MPa for 10 min. High pressure processing was carried out ina prototype ACIP 6000 (ACB, Nantes, France) of 3.5-L capacity and600-MPa maximum working pressure. Water was used as a pressure-transmitting medium. Non-pressurized samples without LPS wereused as control. All treated and control sampleswere kept at 5 °C during35 d of storage. Two trials were carried out.

2.4. LP system

Lactoperoxidase from bovine milk (DMV International Nutri-tionals, Veghel, Holland) was prepared in distilled–deionized water(15 mg/ml) and stored at−40 °C. Lactoperoxidase activity was quan-tified before addition to sliced smoked salmon according to the meth-od of Marshall, Cole, and Bramley (1986) by determination of theoxidation of ABTS (2,2-azinodi-[3-ethylbenzthiazoline-sulphonicacid]) (Sigma-Aldrich, Alcobendas, Spain) at 412 nm and expressed

in ABTSU according to Shindler, Childs, and Bardsley (1976). Potassi-um thiocyanate (KSCN, Merck, Darmstadt, Germany) was prepared ina 1 M aqueous solution and autoclaved at 121 °C for 15 min. Hydro-gen peroxide 30% aqueous solution was obtained from Merck. In theexperiments with the LPS, the enzyme was activated by adding lacto-peroxidase to reach an estimated final activity of 2.8 ABTSU/g, fol-lowed by 40 μl/g of KSCN and 10 μl/g of H202.

2.5. Microbiological analysis

To count the surviving L. monocytogenes in inoculated samples,salmon slices (20 g) were transferred aseptically to a sterile stomacherbag, diluted 10-fold in sterile 0.1% (wt/vol) peptone water solution andhomogenized for 90 s in a Stomacher 400 (A.J. Seward Ltd., London,UK). Decimal solutions were prepared in the same solution and spreadon duplicate plates of PALCAM Listeria Selective Agar (Merck) incubat-ed at 37 °C for 48 h. Total mesophilic counts in non-inoculated salmonsamples (20 g) were determined in duplicate on Tryptic Soy YeastExtract Agar (TSYEA) (Biolife) and incubated at 30 °C for 72 h.

2.6. Physicochemical analysis

pH was measured using a pH meter GLP22 (Crison InstrumentsS.A., Barcelona, Spain). Smoked salmon samples (10 g) were homog-enized with 90 ml of distilled water in stomacher bags for 90 s.Water activity (aw) was examined in 10 g samples with an AqualabSeries 3 water activity meter (Decagon Devices, Inc., Pullman, WA,USA). All measurements were performed in duplicate samples.

2.7. Biogenic amines

Biogenic amines in smoked salmon were extracted in duplicatesamples using the procedure of Krause, Bockhardt, Neckermann,Henle, and Klostermeyer (1995). The quantitative analysis, after de-rivatization with dabsyl chloride, was carried out by reverse phaseHPLC using a System Gold (Beckman Coulter, Palo Alto, CA, USA) liq-uid chromatograph, according to Romero, Gázquez, Bagur, andSánchez-Viñas (2000). All separations were carried out in duplicateon a Waters Novo-Pak C18 column (Water Mildford, MA, USA). A bio-genic amine standard mixture (Sigma-Aldrich) was also analyzed forthe identification and quantification of biogenic amines. Results wereexpressed as mg of biogenic amine per kg of cold-smoked salmon.

2.8. Color and texture

L* (lightness, intensity of white color), a* (+a, red; −a, green)and b* (+b, yellow; −b, blue) values were evaluated using a Chrom-ometer CM-700d (Konica Minolta Ltd., Osaka, Japan) and eight mea-surements were taken from each sample in different locations of thesmoked salmon slices.

Smoked salmon texture was determined using an Instron Univer-sal testing Machine 4301 (Instron Ltd., Barcelona, Spain) controlledby the Bluehill V2.0 software. Hardness (N), considered as the maxi-mum force required to compress the sample, was evaluated with aKramer cell with smoked salmon slices divided into approximately6.5×2×1 cm pieces. The shear strength (N) required to shearthrough the sample was examined with a Warner–Bratzler bladewith samples cut into 5×3×1 cm pieces. Three measurements weretaken from each sample.

2.9. Statistical analysis

Analysis of variance with treatment and time of refrigeration asmain effects was carried out using SPSS Win 12.0 software (SPSSInc., Chicago, IL, USA). The significance of differences between

28 R. Montiel et al. / Innovative Food Science and Emerging Technologies 16 (2012) 26–32

means for the same time of storage was evaluated by Tukey test witha confidence interval of 95%.

3. Results and discussion

3.1. Effect of HHP treatments and LPS on L. monocytogenes

L. monocytogenes H66a counts in control and treated cold-smokedsalmon during refrigerated storage are shown in Table 1. Pathogenlevels were significantly reduced (Pb0.001) by treatment and in-creased (Pb0.001) during refrigerated storage. L. monocytogenesH66a initial counts in inoculated smoked salmon were 6.93 log cfu/gat 0 h and grew to 7.95 log cfu/g after 35 d of refrigeration at 5 °C. Im-mediately after treatment, significant (Pb0.05) reductions achievedwith individual treatments were 2.72 and 1.06 log units at 450 MPaand the LPS, respectively, compared to control samples. An additiveantimicrobial effect was observed when 450 MPa was applied in LPStreated samples, with reductions of 3.84 log units. On the contrary,no antimicrobial activity was detected after HHP at 250 MPa individ-ually or in combination with the LPS. During the refrigerated storageat 5 °C, a regrowth of L. monocytogenes was observed after the initialdecrease. However, after 35 d, levels in cold-smoked salmon treatedat 450 MPa, individually or in combination with LPS, were 0.94 and2.70 log units lower than initial counts, respectively.

Pressurization of cold-smoked salmon at 250 MPa for 20 min didnot inactivate a mixture of four L. monocytogenes strains but increasedlag times 17 and 10 d at 5 °C and 10 °C, respectively (Lakshmanan &Dalgaard, 2004). These results were not confirmed in the presentwork at 250 MPa, with similar L. monocytogenes levels in pressurizedand control smoked salmon throughout the refrigeration period stud-ied. The effect of increasing time of pressurization on the survival ofL. monocytogenes in cold-smoked salmon was low when 450 MPawas assayed at different times (Medina et al., 2009). According tothese authors, pressurization at 450 MPa for 10 min in smoked salm-on attained an FSO (Food Safety Objective) of 2 log cfu/g for a shelf-life of 35 d at 5 °C.

Only a reduction of 1 log was achieved by the LPS addition to cold-smoked salmon in the present work. L. monocytogenes reinitiated itsgrowth from day 2 of refrigerated storage to reach values not signifi-cantly different to those in control samples at the end of refrigeration.The bactericidal activity of the LPS against L. monocytogenes in milkand dairy products has been reported (Arqués et al., 2008; García-Graells et al., 2003; Zapico et al., 1998). Fewer studies on the usethis antimicrobial system in fish and meat products have been pub-lished. LPS added to fresh meat at 1.9 U/cm2 exhibited a bacteriostaticeffect on L. monocytogenes during 7 d at 12 °C, with levels 2.6 logunits lower than the control at the end of the refrigeration period(Elliot et al., 2004). Whey protein films incorporating the LPS reducedinitial counts of L. monocytogenes in smoked salmon by approximate-ly 4 log units during 35 d of refrigeration at 4 °C (Min et al., 2005).According to our results, the initial bactericidal effect of the LPScould be limited by the high initial counts of L. monocytogenes incold-smoked salmon, as low numbers (30–50 cfu/ml) were totally

Table 1L. monocytogenes counts (log cfu/g) in cold-smoked salmon treated with HHP at 250 and 4

0 d 1 d 2 d

Control 6.93±0.04dA 6.98±0.08eA 6.86±0.0250 MPa 6.92±0.10dA 6.98±0.16eA 6.98±0.0LPS 5.87±0.15cA 6.06±0.07cA 6.01±0.0250 MPa+LPS 6.48±0.50dA 6.50±0.27dA 6.81±0.1450 MPa 4.21±0.58bA 4.45±0.13bA 4.06±0.0450 MPa+LPS 3.09±0.22aB 1.97±0.30aA 3.40±0.2

Values are mean±SD of duplicate determinations in two experiments.Means within the same column with different lower-case superscripts differ significantly aMeans within the same row with different upper-case superscripts differ significantly at Pb

inactivated in a semi-synthetic medium, raw cow milk and a buffersolution by this antimicrobial system at 4 °C or 35 °C, whereas whenmedium (104 cfu/ml) or high populations (107 cfu/ml) were present,the LPS failed to cause death of the pathogen (El-Shenawy, García, &Marth, 1990).

HHP at 450 MPa combined with the LPS against L. monocytogenes insmoked salmon resulted in a higher lethality of the pathogen than treat-ments applied individually. The highest rate of inactivation achievedwas 3.84 log units with respect to the control salmon. This reductionwas synergistic mainly at the end of the refrigeration period, as thecombined effect of the two treatments was higher than the sum of re-ductions achieved by 450 MPa and the LPS individually. Synergistic ef-fect of HHP treatments in combination with the LPS against L. innocuaand L. monocytogenes has been probed inmilk. L. innocuawas efficientlyinactivated (≥6 log cfu/ml) at mild pressures (400MPa), whereas inthe absence of LPS, the same pressure caused reductions of approxi-mately 2 log units (García-Graells et al., 2003). Also the combinationof high pressure homogenization (HPH) at 75–130 MPa and the LPSresulted in an immediate synergistic effect on L. monocytogenes inskimmilk (Vannini, Lanciotti, Baldi, & Guerzoni, 2004). Our results con-firm the synergistic antimicrobial effect between HHP and the LPS insmoked salmon refrigerated during 35 d at 5 °C.

3.2. Effect of HHP treatments and LPS on total viable counts

Mean total viable counts (TVC) in control and treated samples ofcold-smoked salmon during refrigerated storage are given in Table 2.TVC in control smoked salmon increased about 5 log units during5 weeks of refrigeration at 5 °C. Bacterial counts were significantly re-duced (Pb0.001) by HHP treatments and the LPS and increased(Pb0.001) during refrigerated storage. Immediately after treatment,only combined treatments significantly reduced TVC in smoked salmon.Moreover, the combination of both treatments exhibited an antimicro-bial synergistic effect against total viable microorganisms, with reduc-tions of 1.81 and >2 log cfu/g by 250 and 450MPa combined with theLPS. During refrigerated storage, TVC increased in cold smoked salmon,with higher levels in samples treated at 250 MPa or the LPS applied in-dividually, whereas they were under the detection limit of 1 log cfu/gwhen 450 MPa and the LPS were applied in combination. At the endof the refrigerated storage, TVC in control cold-smoked salmon werehigher than 8 log cfu/g, and ranged from 6.06 to 7.42 log cfu/g in therest of treated samples, with the exception of the smoked salmon sam-ples treated with the combination of 450 MPa and the LPS, with TVCbelow the detection limit of 1 log cfu/g.

The lag phase of TVC was significantly prolonged in HHP-treatedcold smoked salmon (Lakshmanan & Dalgaard, 2004), as observedin the present work only at the more intense conditions. Similar re-sults were obtained by Medina et al. (2009) when smoked salmonwas pressurized at 450 MPa for 5 to 25 min. LPS added to freshmeat delayed the growth of microorganisms during 42 d of refrigera-tion (Elliot et al., 2004). Also the use of whey protein films incorporat-ing the LPS in smoked salmon reduced TVC about 1 log unit anddelayed the bacterial growth at 4 °C as well as at 10 °C (Min et al.,

50 MPa, the LPS and their combinations, during refrigerated storage at 5 °C.

7 d 20 d 35 d

4dA 7.03±0.19cA 7.31±0.08dB 7.95±0.18cdC

6dA 7.10±0.16cA 8.09±0.02eB 8.50±0.03dC

8cA 6.27±0.21bAB 6.82±0.62cBC 7.36±0.62cC

9dA 6.85±0.07bcA 6.69±0.09cA 7.99±0.11cdB

6bA 4.44±0.30aA 5.94±0.05bB 5.99±0.09bB

8aB 4.34±0.41aC 4.46±0.21aC 4.23±0.00aC

t Pb0.05.0.05.

Table 2Total viable counts (log cfu/g) in cold-smoked salmon treated with HHP at 250 and 450 MPa, the LPS and their combinations, during refrigerated storage at 5 °C.

0 d 1 d 2 d 7 d 20 d 35 d

Control 3.16±0.93bA 4.83±0.01dB 4.24±0.04bAB 5.46±0.35cBC 6.82±0.28bCD 8.24±0.03eD

250 MPa 3.61±0.45bA 4.03±0.02cAB 4.57±0.01bAB 4.90±0.72cB 7.42±0.50bC 7.42±0.12dC

LPS 2.96±0.22bA 4.92±0.03dB 4.86±0.03bB 4.74±0.69cB 6.38±0.03bC 6.80±0.19cC

250 MPa+LPS 1.35±0.86aA 1.68±0.15bA 1.20±0.80aA 1.83±0.14abA 6.70±0.03bB 6.06±0.29bB

450 MPa 2.63±0.80bA 1.90±0.25bA 1.40±0.95aA 1.70±0.07abA 1.67±1.78aA 6.11±0.43bB

450 MPa+LPS b1.00aA b1.00aA b1.00aA b1.00aA b1.00aA b1.00aA

Values are mean±SD of duplicate determinations in two experiments.Means within the same column with different lower-case superscripts differ significantly at Pb0.05.Means within the same row with different upper-case superscripts differ significantly at Pb0.05.

29R. Montiel et al. / Innovative Food Science and Emerging Technologies 16 (2012) 26–32

2005). In the present work, the initial bactericidal effect of LPS on TVCwas not observed, although this natural antimicrobial reduced thebacterial growth rate.

A combination of several preservation methods may providegreater protection than a single method alone, improving the safetyand quality of the food (Deegan, Cotter, Hill, & Ross, 2006). Synergis-tic effect of LPS and HHP treatment against Gram-positive and Gram-negative bacteria has been reported (García-Graells et al., 2003). Inthe present work, as a result of the addition of LPS in smoked salmon,total viable microorganisms were efficiently inactivated at 450 MPafor 10 min, remaining under detectable levels during the 35 d of re-frigeration at 5 °C. Both preservation treatments may be applied incombination to extend the shelf-life of cold-smoked salmon at refrig-eration temperatures.

3.3. pH and aw

The pH values during the refrigerated storage of control and trea-ted cold-smoked salmon with HHP and LPS, individually or in combi-nation, are shown in Table 3. The treatments (Pb0.001) and storagetime (Pb0.001) influenced significantly the pH of the product. Atday 0, the pH in control smoked salmon was 6.63 and ranged from6.29 to 6.55 in samples treated with HHP, the LPS or their combina-tion. At the end of the refrigeration, pH values in the range of 6.19to 6.33 did not differ significantly.

Water activity values during the refrigerated storage of controland treated smoked salmon with HHP and the LPS, individually orin combination, are given in Table 3. The treatments (Pb0.001) andstorage time (Pb0.001) influenced significantly aw values. On day 0,

Table 3Values of pH and aw in cold-smoked salmon treated with HHP at 250 and 450 MPa, theLPS and their combinations, during refrigerated storage at 5 °C.

0 d 20 d 35 d

pHControl 6.63±0.06cC 6.36±0.06bB 6.24±0.01aA

250 MPa 6.53±0.06bcB 6.42±0.04bB 6.16±0.18aA

LPS 6.55±0.15bcB 6.23±0.02aA 6.19±0.05aA

250 MPa+LPS 6.29±0.06aB 6.26±0.02aAB 6.19±0.03aA

450 MPa 6.38±0.05abA 6.36±0.05bA 6.33±0.01aA

450 MPa+LPS 6.34±0.14abA 6.35±0.01bA 6.32±0.02aA

awControl 0.951±0.001bA 0.950±0.003aA 0.950±0.007aA

250 MPa 0.962±0.003cB 0.952±0.001aA 0.956±0.006abAB

LPS 0.946±0.002aA 0.952±0.002aA 0.949±0.004aA

250 MPa+LPS 0.965±0.001cC 0.955±0.002aA 0.961±0.002bcB

450 MPa 0.963±0.001cA 0.963±0.003bA 0.960±0.001bA

450 MPa+LPS 0.976±0.001dB 0.969±0.001cA 0.970±0.004cA

Values are mean±SD of duplicate determinations in two experiments.Means within the same column with different lower-case superscripts differ signifi-cantly at Pb0.05.Means within the same row with different upper-case superscripts differ significantlyat Pb0.05.

values of aw in samples treated with the LPS were lower (Pb0.05)than in control salmon, whereas higher (Pb0.05) values were ob-served with the rest of treatments. During refrigeration, higher valuesof aw tended to be detected in samples pressurized at 450 MPa, aloneor in combination with the LPS. This increase was also observed(Montiel, De Alba, Bravo, Gaya, & Medina, 2012) in pressurizedsmoked cod.

3.4. Biogenic amines

Biogenic amine content during the refrigerated storage of controland treated smoked salmon is given in Table 4. All biogenic amines test-ed in smoked salmon, except tryptamine, were significantly affected bytreatments (Pb0.001) and storage time (Pb0.001). Histamine was notdetected in cold-smoked salmon throughout the refrigeration periodof 35 d at 5 °C. Tryptamine was not detected in control smoked salmonat day 0, phenylethylamine, putrescine, tyramine, spermidine and sper-mine valueswere lower than 4 mg/kg, whereas cadaverine content was21.61 mg/kg. At the end of the storage, tryptamine was not detected incontrol or treated smoked salmon and spermidine and spermine con-tents were lower than 2 mg/kg. Tyramine and cadaverine exhibitedvalues of 128.75 and 214.35 mg/kg, respectively, in control smokedsalmon, and their content was similar in HHP-treated samples at250 MPa. The rest of the treatments reduced, in general, the formationof these two biogenic amines, which was avoided by the combinationof HHP and LPS during 35 d of storage at 5 °C.

Biogenic amines are important indicators of spoilage in vacuum-packed cold-smoked salmon. Salmon has a high content of nonpro-tein nitrogen, mainly free amino acids, and consequently, the produc-tion of histamine and other biogenic amines during storage is possible(Jørgensen et al., 2000). Cadaverine, histamine, putrescine and tyra-mine were detected during the chilled storage of smoked salmonwith high density of microorganisms (Cantoni, Moret, & Comi,1993), and putrescine and tyramine were formed in smoked salmontreated at 200 MPa and stored at 14 °C (Lakshmanan & Dalgaard,2004). In the present work, high concentrations of cadaverine and ty-ramine were detected at the end of refrigeration in control and pres-surized at 250 MPa smoked salmon samples with TVC higher than 7log units. Diamines such as putrescine or cadaverine may boost thetoxicity of tyramine (Bover-Cid & Holzapfel, 1999), increasing therisk of smoked salmon refrigerated during large periods of time.

According to our results, HHP at 450 MPa in combination with theLPS was the most effective treatment avoiding biogenic amine forma-tion, with levels lower than 5 mg/kg after 35 d at 5 °C. This low amountof biogenic amines in smoked salmon observed in the present workcould be attributed to the low levels of total bacterial counts whichremained under the detection limit during all the refrigerated storage.

3.5. Color

Changes in color parameters (L*, a* and b*) of smoked salmon arepresented in Table 5. L* (lightness or brightness) increased signifi-cantly with treatments (Pb0.001) and storage time (Pb0.001). After

Table 4Biogenic amines (mg/kg) in cold-smoked salmon treated with HHP at 250 and450 MPa, the LPS and their combinations, during refrigerated storage at 5 °C.

0 d 20 d 35 d

TRN Control 0.00±0.00aA 0.65±0.76aA 0.00±0.00aA

250 MPa 4.62±3.39bB 0.00±0.00aA 0.00±0.00aA

LPS 0.00±0.00aA 0.00±0.00aA 0.00±0.00aA

250 MPa+LPS 0.00±0.00aA 1.63±1.88aA 0.00±0.00aA

450 MPa 0.00±0.00aA 5.34±6.19aA 0.00±0.00aA

450 MPa+LPS 0.86±1.01aA 0.00±0.00aA 0.00±0.00aA

FN Control 0.18±0.21aA 17.34±11.27bB 19.15±8.73bB

250 MPa 7.94±0.28bA 6.87±1.27aA 15.99±3.26bB

LPS 0.00±0.00aA 0.00±0.00aA 0.32±0.37aA

250 MPa+LPS 0.00±0.00aA 0.53±0.43aB 0.00±0.00aA

450 MPa 0.69±0.56aA 2.46±1.08aA 2.35±2.72aA

450 MPa+LPS 0.38±0.44aA 0.00±0.00aA 0.00±0.00aA

PUT Control 0.50±0.59aA 9.06±8.64bA 25.56±10.65bB

250 MPa 4.65±1.06bAB 2.37±0.39abA 9.04±4.47aB

LPS 0.96±0.82aA 0.52±0.35aA 1.78±2.06aB

250 MPa+LPS 0.21±0.06aA 1.02±0.23aB 0.00±0.00aA

450 MPa 1.45±0.39aA 1.14±0.23abA 0.82±0.92abA

450 MPa+LPS 1.15±1.09aA 0.04±0.07aA 0.00±0.00aA

CAD Control 21.61±0.12bA 288.83±78.38cB 214.35±77.95bB

250 MPa 109.34±5.42dA 152.88±26.58bA 245.10±58.76bB

LPS 14.51±3.22bA 10.73±10.59aA 35.33±13.43aB

250 MPa+LPS 0.90±0.12aA 2.29±0.15aB 2.67±0.07aC

450 MPa 32.70±2.38cA 49.44±52.08aA 120.52±33.35abA

450 MPa+LPS 5.26±1.06aB 5.24±1.47aB 2.85±0.14aA

TYR Control 3.99±0.41bA 119.99±9.34cB 128.75±31.37cB

250 MPa 14.11±0.81cA 36.84±9.43bA 102.44±26.19bcB

LPS 4.73±1.88bA 32.01±21.74bA 79.71±21.26bB

250 MPa+LPS 0.00±0.00aA 1.58±0.12aA 28.07±3.42aB

450 MPa 3.29±0.52bA 3.50±2.13aA 4.13±3.38aA

450 MPa+LPS 0.96±0.25aB 0.10±0.11aA 0.62±0.38aAB

SDN Control 0.48±0.18aA 1.59±0.71bcB 0.27±0.28aA

250 MPa 3.04±0.29cB 0.82±0.13abA 0.46±0.36aA

LPS 0.77±0.16aA 0.75±0.10abA 0.53±0.10aA

250 MPa+LPS 1.00±0.11abB 1.96±0.41cC 0.27±0.34aA

450 MPa 0.97±0.27abA 2.44±0.50cB 0.64±0.11aA

450 MPa+LPS 1.37±0.34bB 0.23±0.08aA 0.40±0.04aA

SPN Control 1.18±0.38aA 2.38±0.12bB 0.87±0.46abA

250 MPa 6.59±0.42dC 2.05±0.04abB 1.20±0.55abA

LPS 1.80±0.26abB 1.71±0.05abB 1.00±0.18abA

250 MPa+LPS 2.49±0.16bcB 3.78±1.13cB 0.53±0.35aA

450 MPa 2.19±0.62bcA 4.94±0.92cB 1.55±0.60bA

450 MPa+LPS 3.12±0.54cB 0.76±0.16aA 0.81±0.11abA

Values are mean±SD of duplicate determinations in two experiments.TRN, FN, PUT, CAD, HIS, TYR, SDN and SPN are tryptamine, phenylethylamine, putres-cine, cadaverine, histamine, tyramine, spermidine and spermine, respectively.Means within the same column with different lower-case superscripts differ signifi-cantly at Pb0.05.Means within the same row with different upper-case superscripts differ significantlyat Pb0.05.

Table 5Color characteristics of cold-smoked salmon treated with HHP at 250 and 450 MPa, theLPS and their combinations, during refrigerated storage at 5 °C.

0 d 20 d 35 d

L*Control 40.67±2.87aA 40.33±1.94aA 43.61±2.21aB

250 MPa 49.83±1.68cA 50.44±1.88cA 52.06±1.61cB

LPS 43.74±2.08bA 44.45±2.79bAB 46.19±2.21bB

250 MPa+LPS 51.81±1.36cA 59.42±1.37dC 56.46±2.49dB

450 MPa 61.79±1.62dB 52.53±4.84cA 60.63±1.42eB

450 MPa+LPS 64.31±1.82eA 65.77±1.29eB 65.37±1.38fAB

a*Control 18.42±1.61aA 19.61±1.21cA 21.32±2.71cB

250 MPa 21.57±1.62bA 22.76±1.38dAB 23.42±1.64cB

LPS 17.86±1.67aA 17.35±1.72abA 18.05±1.67bA

250 MPa+LPS 18.42±1.50aB 22.90±1.39dA 15.57±2.92aC

450 MPa 21.42±2.02bB 18.04±2.06bcA 22.34±1.40cB

450 MPa+LPS 17.47±2.45aA 15.90±2.16aA 17.78±2.04bA

b*Control 13.26±2.57bA 13.19±2.20abA 14.21±2.66bA

250 MPa 13.93±2.89bB 13.97±1.67bB 14.49±2.49bB

LPS 12.39±1.95abAB 11.89±1.76aA 13.56±1.79abB

250 MPa+LPS 10.90±1.97aA 13.40±2.20abB 11.77±1.79aAB

450 MPa 13.23±1.51bA 13.32±2.24abA 12.94±1.91abA

450 MPa+LPS 13.69±1.01bA 13.58±1.04abA 13.48±1.34abA

Values are mean±SD of eight determinations in two experiments.Means within the same column with different lower-case superscripts differ signifi-cantly at Pb0.05.Means within the same row with different upper-case superscripts differ significantlyat Pb0.05.

30 R. Montiel et al. / Innovative Food Science and Emerging Technologies 16 (2012) 26–32

treatments, L* values varied from 40.67 in control samples to 64.31when the LPS was added to smoked salmon in combination withHHP at 450 MPa, with lower differences when the LPS was applied in-dividually. At the end of refrigeration at 5 °C, these differences weremaintained, resulting in a brighter and less transparent appearanceof the muscle in all the treated samples with respect to controlsmoked salmon.

The values of a* (redness) were significantly affected by treat-ments (Pb0.001) whereas they were not changed by time of storage.Immediately after treatment, redness values were significantly(Pb0.05) higher in HHP-treated samples applied individually thanin control salmon. At the end of the refrigeration, these samples to-gether with control salmon exhibited the higher (Pb0.05) values forredness.

The values of b* (yellowness) were significantly affected by treat-ments (Pb0.001) but not by time of storage. Immediately after treat-ment, yellowness decreased (Pb0.05) when LPS was added tosmoked salmon alone or in combination with HHP at 250 MPa. Dur-ing the refrigeration, differences in b* values between control andtreated cold-smoked salmon diminished.

Color is considered as one of the most important attributes of foodappearance. Changes in color parameters by HHP have been observedat pressures higher than 150–200 MPa in different seafood species(Matser, Stegeman, Kals, & Bartels, 2000), and higher L* and lowera* values were associated with fresh salmon pressurization over200 MPa (Amanatidou et al., 2000). These changes have been attrib-uted to denaturation of myofibrillar and sarcoplasmic proteins(Matser et al., 2000). Free amino acid content was affected by highpressure treatments in smoked fish, and these changes were associat-ed (Erkan et al., 2011) with changes in color.

Few studies about the effect of LPS, alone or in combination withpressure, on food color have been published. The use of whey proteinfilms incorporating this antimicrobial system did not affected thecolor parameters in smoked salmon (Min et al., 2005). According toour results, L*, a* and b* values were affected by HHP and LPS record-ing a lightness increase in smoked salmonwith all treatments assayedin the present work, whereas the impact of the treatments on rednessand yellowness was lower.

3.6. Texture

Changes in texture parameters (hardness and shear strength) ofcontrol and treated smoked salmon during storage at 5 °C are pre-sented in Table 6. HHP and LPS significantly increased (Pb0.001)the hardness of smoked salmon determined with the Kramer cell.After the treatment, significantly higher (Pb0.05) hardness valueswere registered in smoked salmon treated at 250 MPa in combinationwith the LPS and at 450 MPa, individually and in combination withthe LPS compared with untreated samples. At the end of the refriger-ation, hardness values in smoked salmon pressurized at 450 MPawere higher (Pb0.05) than in control samples. Pressurization andstorage time also affected (Pb0.001) the shear strength of cold-smoked salmon measured using the Warner–Bratzler blade, withvalues tending to increase significantly (Pb0.05) in treated with re-spect to control smoked salmon.

Table 6Texture characteristics of cold-smoked salmon treated with HHP at 250 and 450 MPa,the LPS and their combinations, during refrigerated storage at 5 °C.

0 d 20 d 35 d

Hardness (N)Control 70.42±14.56aA 121.83±23.65aB 79.82±17.28aA

250 MPa 91.28±27.66aA 130.36±42.21aA 90.21±12.68abA

LPS 103.58±24.73aA 148.32±19.16aB 153.51±42.89abB

250 MPa+LPS 235.33±67.33bA 195.59±39.54abA 183.92±80.95abcA

450 MPa 236.33±96.31bA 319.84±131.33bA 222.63±48.28bcA

450 MPa+LPS 268.30±87.22bA 302.95±156.55bA 309.53±164.22cA

Shear strength (N)Control 6.17±1.13aA 8.37±3.14aA 8.44±1.63aA

250 MPa 7.27±1.47abA 7.56±3.15aB 9.85±2.02aA

LPS 11.73±4.61cA 29.07±14.39bB 15.38±6.50abAB

250 MPa+LPS 7.98±1.52aA 15.93±10.30abAB 22.97±15.8bB

450 MPa 11.68±3.89cA 10.79±2.60aA 17.39±4.43abB

450 MPa+LPS 11.07±1.54bcA 16.48±5.31abB 14.91±1.30abAB

Values are mean±SD of three determinations in two experiments.Means within the same column with different lower-case superscripts differ signifi-cantly at Pb0.05.Means within the same row with different upper-case superscripts differ significantlyat Pb0.05.

31R. Montiel et al. / Innovative Food Science and Emerging Technologies 16 (2012) 26–32

The texture is an important characteristic which determines thequality of fish and the consumer acceptance. During chilled storageof seafood products, degradation processes occur, mostly carried outinitially by endogenous muscle enzymes and later by microbial en-zymes, reducing the quality (Lakshmanan, Patterson, & Piggott,2005; Lakshmanan, Piggott, & Paterson, 2003; Ortea, Rodríguez,Tabilo-Munizaga, Pérez-Won, & Aubourg, 2010). HHP technologyhas been proposed to delay the enzymatic degradation of seafoodproducts during chilled storage. Although an increase of proteolysishas been registered in cod as a consequence of HHP treatments at200 MPa (Angsupanich & Ledward, 1998), a decrease of the autolyticactivity has been observed in octopus pressurized at 450 MPa(Hurtado, Montero, Borderías, & Solas, 2001). The effect of pressureon the textural characteristics differed among various seafood prod-ucts (Gómez-Estaca et al., 2007; Hurtado et al., 2001), recording ingeneral a hardness increase in smoked salmon after pressurization(Lakshmanan, Miskin, & Piggott, 2005). Less known is the effect ofLPS on food texture. This enzymatic system added to Kasar cheeseresulted in a firmness increase after 60 and 90 d of ripening(Atamer et al., 1999). In the present work, hardness and shearstrength values tended to increase when 450 MPa was applied aloneor in combination with the LPS.

4. Conclusions

HHP treatments at 450 MPa for 10 min in combination with the LPSincreased synergistically the antimicrobial effect against L. monocytogenesin cold-smoked salmon. This combined treatment avoided the pathogenrecovery observedwhen individual treatmentswere applied and delayedthe spoilage of smoked salmonmaintaining total viable counts belowthe detection limit during 35 d of storage at 5 °C. Changes in qualityparameters were related with the increase of L* values and firmnessof smoked salmon muscle. The lactoperoxidase system added tosmoked salmon in combination with high pressure treatments at450 MPa for 10 min might be used as a hurdle technology approachagainst L. monocytogenes, increasing the safety and shelf-life ofcold-smoked salmon.

Acknowledgments

This work has received support from projects AGL2007-65235-C02-01, AGL2010-16600 and CSD2007-00016 (Ministry of Science

and Innovation). The authors thank Buenaventura Rodríguez andMáximo de Paz for their valuable help in treatments.

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