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Title: Effect of high pressure processing on Listeria spp. and on the textural andmicrostructural properties of cold smoked salmon
Authors: Birna Gudbjornsdottir, Asbjorn Jonsson, Hannes Hafsteinsson, Volker Heinz
PII: S0023-6438(09)00242-4
DOI: 10.1016/j.lwt.2009.08.015
Reference: YFSTL 2283
To appear in: LWT - Food Science and Technology
Received Date: 6 January 2008
Revised Date: 5 August 2009
Accepted Date: 21 August 2009
Please cite this article as: Gudbjornsdottir, B., Jonsson, A., Hafsteinsson, H., Heinz, V. Effect of highpressure processing on Listeria spp. and on the textural and microstructural properties of cold smokedsalmon, LWT - Food Science and Technology (2009), doi: 10.1016/j.lwt.2009.08.015
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LWT- FOOD SCIENCE AND TECHNOLOGY 1
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Title: Effect of high pressure processing on Listeria spp. and on the textural and 4
microstructural properties of cold smoked salmon 5
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Authors: Birna Gudbjornsdottir*, Asbjorn Jonssona, Hannes Hafsteinssona, Volker Heinzb 7
Authors address: 8
aMatis ohf , Skúlagata 4, 101 Reykjavik, Iceland 9
bGerman Institute of Food Technology (DIL e.V.), Professor-von-Klitzing-Straße 7, D-49610 10
Quakenbrück, Germany 11
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*Corresponding author: Birna Gudbjornsdottir, Bollagarðar 33, 170 Seltjarnarnes, Iceland, 13
Tel: +354 5611257, Mobile phone: +354 8684258. 14
E-mail address: [email protected] 15
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Runnning title: Effect of high pressure processing in reducing Listeria 17
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Abstract 20
Investigation of the effect of high pressure processing (HPP) at very short time on the 21
inactivation of Listeria innocua was conducted as well as the effect on texture and 22
microstructure. Lipid oxidation, colour and background bacterial flora was studied as well. 23
HPP at 700-900 MPa for 10 s increased the inactivation of L. innocua in cold smoked salmon 24
from 4500 cfu/g to non detectable level (<0.3 cfu/g). L. innocua was more sensitive to HPP 25
than the background flora tested. The product presented good microbiological quality and 26
there was no indication of lipid oxidation. The effect of HPP on the redness of the product 27
was not observed, however immediate effect on the lightness was noticed and the salmon 28
becomes lighter in colour as a function of both time and pressure. The effects on the 29
microstructure increased with both time and pressure and were most significant at 900 MPa 30
and 60 s. The effect on microstructure coincides with the reduction of the bacteria. The 31
knowledge from this study provides information for the industry on the development of HPP 32
at 400-900 MPa with short pressure time of less than 60 s. 33
Keywords: Smoked salmon, microstructure, high pressure, Listeria, shelf life 34
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Introduction 42
Global aquaculture production has increased in recent years and is now growing more rapidly 43
than all other animal food production sectors. A great proportion of farmed Atlantic salmon 44
reaches the worldwide market as a cold smoked product, but smoking is one of the oldest 45
processing methods that have been used to extend the shelf life of food. While the world 46
production increases, problems related to bacterial contamination in cold smoked salmon still 47
persist (Gram, 2001; Gombas, Chen, Clavero, & Scott, 2003; Gudmundsdottir, 48
Gudbjornsdottir, Lauzon, Einarsson, Kristinsson & Kristjansson, 2005). Due to the fact that 49
the temperature during the smoking never exceeds 28 °C it does not have any significant 50
effect on Listeria spp. Many processing parameters have been tested to minimise the 51
occurrence of L. monocytogenes, such as combinations of NaCl and low temperature, other 52
preservatives (e.g. diacetate and lactate) or by the use of bioprotective cultures that can inhibit 53
growth at refrigerated temperatures (Huss, Jørgensen & Vogel, 2000; Gram, 2001; Yoon, 54
Burnette, Abou-Zied, & Whiting, 2004, Fonnesbech, Yin, Hyldig, Mohr, & Gram, 2006; 55
Tomé, Teixeira, & Gibbs, 2006). However these methods could not prevent the growth of this 56
bacterium. If the organism cannot be eliminated and growth-inhibiting steps are not 57
introduced, the hazard has to be controlled by limiting shelf life (at 4 °C) to ensure that no 58
more than 100 cells/g are present at the time of consumption. Time limits for storage may 59
need to be established by each processor because it should reflect the initial level of the 60
organism in freshly produced product. To meet demands from consumer about safe food with 61
prolonged shelf life, it is necessary to develop a new processing method for smoked salmon. 62
Over the last decade, investigators have explored the possibilities of applying some novel 63
technologies against this pathogen. High pressure processing (HPP) is one of these promising 64
technologies. It is a non-thermal preservation technique that depends on pressure, time, 65
temperature and product characteristics and it allows micro-organisms to be inactivated with 66
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fewer changes in texture, colour and flavour as compared to conventional technologies 67
(Knorr, 1993; Carpi, Gola, Maggi, Rovere & Buzzoni, 1995; Cheftel, 1995; Torres & 68
Velazquez, 2005). 69
High pressure processing (HPP) of foods was first reported by Hite (1899) who used this 70
technology to increase the shelf life of milk. Since then several studies on different food items 71
have been published. Most of the studies related to the application of HPP to seafood have 72
been conducted on its effect on proteins (Angsupanich, Edde, & Ledward, 1999), muscle 73
color (Ohshima, Ushio, & Koizumi, 1993; Amanatidou, Schlüter, Lemkau, Gorris, Smid, & 74
Knorr, 2000), lipids (Ohshima, Nakagawa, & Koizumi, 1992; Chevalier, Bail, & Ghoul, 75
2001) and bacteria (Smelt, 1998; Amanatidou et al., 2000). The effect of HPP on L. 76
monocytogenes has been intensively studied regarding the effect of treatment time (Patterson, 77
Quinn, Simpson, & Gilmour, 1995; Simpson & Gilmour, 1997), of pressure (Shigehisa, 78
Ohmori, Saito, Taji, & Hayashi, 1991) and the minimum conditions of the three parameters 79
(pressure, time, temperature) to maximize the reduction of cell viability (Ritz, Jugiau, Rama, 80
Courcoux, Semenou & Federighi, 2000). Lakshmanan & Dalgaard (2004) showed that 81
pressure at 250 MPa did not inactivate L. monocytogenes but lag phases of 17 and 10 days 82
were observed at 5 and 10 °C, respectively. Pressure at 200 MPa had a marked effect on both 83
the colour and the texture of chilled cold-smoked salmon. Another study showed that high-84
pressure treatment of salmon paste extended shelf life from 60 to 180 days at 3 or 8 °C 85
without significant chemical, microbiological, or sensory changes and completely inactivated 86
pathogens present in the inoculated sample (Carpi et al., 1995). Montero, Gomez-Estaca and 87
Gomez-Guillen (2007) presented that cold smoked dolphinfish processed under severe salting 88
and smoking conditions (2.93 % salt and 82 ppm phenol) in combination with pressurization 89
at 300 MPa at 20°C for 15 min kept L. monocytogenes counts under the detection limit 90
throughout 100 days of storage. The resistance of microorganisms to pressure varies 91
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considerably and mainly depends on pressure, time and temperature. By increasing the 92
pressure and time of treatment the number of L. monocytogenes in hard cheese, meat products 93
and fruit juice decreased proportionally (Fonberg-Broczek, Windyga, Szczawinski, 94
Szczawinska, Pietrzak & Prestamo, 2005). L .monocytogenes has been shown to be very 95
sensitive to pressure changes and due to the cost of HPP it would be desirable to increase 96
treatment pressure and keep the treatment time short (Chen, Guan & Hoover, 2006). Most of 97
the studies related to application of HPP of seafood apply 200 to 700 MPa pressure for 3, 5, 98
10, 15 or 20 minutes (Torres &Velazquez, 2005). 99
However, new development in high pressure technology enables high pressure to be reached 100
in 10s. The objective of this research was to study the effect of HPP (400 to 900 MPa) on the 101
survival of Listeria innocua and the characteristics (microstructure, texture and colour) of 102
cold smoked salmon during 10, 20, 30 and 60 s. The changes in counts of total viable 103
psychrotrophic bacteria (TVC), lactic acid bacteria (LAB) and Bacillus spores (PCA) were 104
investigated. 105
2. Materials and Methods 106
2.1. Preparation of bacterial culture 107
In order to decide which strain should be selected for this study a pre-study was performed on 108
six strains of L. monocytogenes and 2 strains of L. innocua. All strains were obtained from 109
IFL-collection after isolation from product or processing environment during cold smoking. 110
The strains were cultivated overnight in Tryptic Soy Broth supplemented with 0.6 g Yeast 111
Extract/100 g (Difco) at 35 °C and subcultured twice. All these strains have been compared 112
by a genetic typing technique using pulsed-field gel electrophoresis (Gudmundsdottir et al., 113
2005). The following strains were tested for the efficacy of HPP on reducing the bacterial 114
number: Listeria innocua, strains EU2173/E-34 and EU2172/E-33; Listeria monocytogenes, 115
strains E1, E5, H-01-170-2, L-327, L-435 and L-462. 116
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2.2. Raw material and sample preparation. 117
Cold smoked salmon (CSS). 118
Atlantic salmon (Salmo salar) was farmed and slaughtered at Rifos hf. on the north coast of 119
Iceland. A sample of 50 fishes of 3 to 4 kg each was randomly selected from a large 120
population that was slaughtered that day and kept on ice. Two days later the salmon was 121
filleted and cold smoked at Reykofninn smokehouse, which is located in the area of 122
Reykjavik. The fillets were salted in brine, containing 8 g NaCl in 100 mL of water for 24 123
hours, and smoked at 24 hours at 18-20 °C. Each fillet was vacuum packed and delivered 124
immediately to the Icelandic Fisheries Laboratory (IFL). The following day the fillets were 125
cut into 30 to 50 g pieces (4 cm x 6 cm) and vacuum packed by Magic VacTM Champion. 126
Before the packaging, one half of the samples were contaminated (spiked) with 1 ml of 2 x 127
105 cfu/mL of bacterial suspension (L. innocua, E-34, the most resistance bacteria from the 128
pre-study) to obtain a final concentration of 103-104 cfu/g in the salmon sample. Dilution was 129
made in a Butterfields buffer. The other half were not contaminated, but high-pressure 130
processed and used for textural and microstructural analysis. The day after (day 5 from 131
slaughter) the samples were transported to Berlin University of Technology, Department of 132
Food Biotechnology and Food Process Engineering. On day 6 and 7 the smoked salmon 133
samples were processed with high-pressure at 400, 500, 600, 700, 800 and 900 MPa for 10, 134
20, 30 and 60 s. On day 8 the samples were transported back to Iceland where they were 135
examined for microbiology at IFL and texture and microstructure at the Technical Institute of 136
Iceland (IceTec). The temperature during this transportation was documented with a logger 137
and the average temperature was 4 °C (data not shown). Water content, water activity, lipid 138
(fat) content, pH, NaCl content and Thiobarbituric acid (TBARS) value were determined to 139
characterize the material used for this study. Samples for these analyses were taken from the 140
raw material and at different steps throughout the process. 141
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142
2.3. Water -, fat- and salt content, lipid oxidation, water activity and pH analysis 143
Water content was measured according to ISO 6496 (1999). The sample was heated in an 144
oven at 103 °C +/- 2 °C for four hours. Water content corresponds to the weight loss. 145
Total fat was determined by extraction with petroleum ether, boiling range 40 °C to 60 °C 146
using an extraction apparatus 2050 Soxtec Avanti Automatic System (AOCS 1998). 147
Salt content was measured according to AOAC (2000). Soluble chloride was extracted from 148
the sample with water containing nitric acid. The chloride content of the solution was titrated 149
with silver nitrate and the end point was determined potentiometrically. 150
Lipid oxidation. Thiobarbituric acid reactive substances (TBARS) were determined by a 151
modified version (Sørensen & Jørgensen, 1996) of the extraction method described by 152
Vyncke (1970, 1975) with few modifications. The sample size was reduced to 15 g and 153
homogenized with 30 ml of 7.5 g/100g of trichloroacetic acid solution containing 0.1 g/100g 154
of both propyl gallate and EDTA. The absorbance of samples and standards were measured at 155
530 nm. TBARS, expressed as µmol malondialdehyde per kilogram of sample (μmol 156
MDA/kg), was calculated using malondialdehyd-bis-(diethyl acetate) as standard. 157
Water activity was measured by using Aw - Wert - Messer (Durotherm) capsule at 22°C and 158
kept in an incubator for at least four hours before a reading was taken. Calibration and 159
temperature corrections were made according to the manufacturer’s instructions. 160
pH analysis. Approximately five grams of minced tissue was mixed with the same amount of 161
water (weight), and pH measurement was made using the PHM 80 Portable Radiometer 162
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Analytical Copenhagen, with an immersed electrode according to instructions of the 163
manufacturer’s manual. 164
All analysis was done in duplicate or triplicate. 165
2.4. Microbial analysis 166
For the microbial analysis 25 g of the salmon samples were added to 225 ml of Maximum 167
Recovery Diluent (MRD, Difco) and mixed in stomacher (Stomacher 400, A. J. Seward, 168
London, UK) for 2 min. Two additional 10-fold dilutions were prepared by adding 1 ml of the 169
previous dilution to 9 ml of MRD. Total viable psychrotrophic count (TVC) was determined 170
by spread plating on Modified Long and Hammer (LH) agar according to van Spreekens 171
(1974) with 1 g NaCl/100g (15°C for 5-7 days). Lactic acid bacteria (LAB) were determined 172
by spread plating on Nitrite Actidione Polymyxin (NAP) agar for 22 °C for 5 days (Modified 173
from: Davidson & Cronin (1973). To confirm existence of LAB, catalase test was performed. 174
The most probable number method (MPN) was used to enumerate Listeria and the media used 175
and steps were according to USDA-FSIS method (2002). Listeria enrichment broth base 176
(UVM formulation, Oxoid) was used as the pre-enrichment step followed by inoculation to 177
Fraser broth (Oxoid) with a subcultured to a plate of Modified Oxford Agar (Difco) from all 178
black tubes. In this case the 3-tube 3 dilution (3 x 3) format was used. The detection limit for 179
this method is MPN 0.3 CFU/g or 1 cfu/100 ml (solid or liquid sample). One millilitre of the 180
homogenated sample was transferred into the first of three test tubes, each containing 10 ml of 181
UVM in double concentration. The next three consecutive dilutions were made in single 182
concentration of UVM. This allowed us to achieve sampling sizes of 1, 0.1 and 0.01 g of 183
salmon per 10 ml. For Bacillus spore count, 10 ml of the 1/10 mixture were heated at 75°C for 184
30 min. The pour plate technique was done on Plate Count Agar (Difco). Plates were 185
incubated at 35 °C for 2 days. All analyses were done in duplicate. 186
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2.5. High pressure processing (HPP) 187
Efficacy of HPP on reducing Listeria spp. (pre-study) 188
The pre-study of HPP was carried out at IceTec using an Autoclave Engineering (Erie, PA, 189
USA) high pressure system. Maximum design pressure for the system was 500 MPa at room 190
temperature. The size of the sample holder was 2.4 L. Pressure transmitting medium was 5 191
ml/100 ml oil (Kutwell 42 from Exxon) in water solution. Cell suspensions (1 ml) inoculated 192
to a gauze were placed in a plastic bag and vacuum packed by Magic VacTM Champion. The 193
bacterial strains were high-pressure treated at 350 MPa for 5 and 20 min at 22 °C. A total of 194
five minutes were needed to reach 350 MPa whereas pressure decompression took 30 s. 195
High-pressure processing of smoked salmon 196
High-pressure processing of smoked salmon was carried out at The Berlin University of 197
Technology, Department of Food Biotechnology and Food Process Engineering, Berlin, 198
Germany, in a designed and constructed lab-scale high pressure system (High Pressure 199
Research Center, Unipress Equipment Division, Sokolowska 29/37, Warsaw, Poland). 200
Maximum design pressure for the system was 1000 MPa at an operating temperature range of 201
–25 °C to 100 °C. The volume of the sample holder was 0.75 L. A mixture of water and 202
glycol (propylene glycol 1.2 propanediol; 50:50) was used as a pressure transmitting medium. 203
The most HPP-resistance strains (L. innocua (E-34)) from the pre-study were chosen to 204
continue the main study. Spiked and unspiked vacuum packed salmon samples were high-205
pressure processed at 400, 500, 600, 700, 800 and 900 MPa pressure for 10, 20, 30 and 60 s. 206
Temperature during holding time (after adiabatic heating) did reach 42 °C in all trials. 207
Unpressurized samples were used as a control. Ten s were needed to reach 400, 500 and 600 208
MPa and 20 s to 25 s to reach 700, 800 and 900 MPa. 209
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2.6. Storage trials with spiked samples of cold smoked salmon 210
The storage trial consisted of samples after two HPP treatments (500 and 900 MPa) and of 211
untreated samples used as a control. The storage temperature was 5.5 °C. The samples were 212
tested for microbial changes (TVC, LAB and Bacillus spores), the survival of L. innocua and 213
for lipid oxidation on days 5, 12, 26 and 41. 214
2.7. Microstucture 215
Samples for microstructure analysis were collected from the HPP treated salmon pieces using 216
a cork knife, 11 mm in diameter. They were embedded in plastic tubes 15 mm in diameter, 30 217
mm length (Kartell, Novigilo, Italy) containing optical coherence tomographical (OCT) 218
compound (embedding medium) (Tissue Tek, Sakura, Torrance, CA) and frozen in liquid 219
nitrogen. Freezing (below –80°C) occurred in approximately 40 s. The frozen specimens were 220
stored at –80 °C until cryosectioning and staining. The specimens were sectioned frozen at –221
27 °C in a Leica CM1800 cryostat (Leica, Heidelberg, Germany) for transverse cuts, 10 μm 222
thick. Sections were mounted on glass slides and stained using Orange G (0.5 g CI 16230 223
(Polysciences, Warrington, PA), 99.0 mL distilled water, 1.0 mL acetic acid). The sections 224
were washed with distilled water and stained for 5 min. in methyl blue solution (0.07 g CI 225
42780 (Sigma, St. Louis, MO), 99.0 mL water and 1.0 mL acetic acid). The stained samples 226
were washed for five minutes with distilled water, dried at room temperature and mounted 227
with MOUNTEX (Histolab, Göeteburg, Sweden). The samples were examined in an optical 228
microscope, Leica DM RA2 at 100 x magnification and images captured using a Leica 229
DC300F digital camera mounted on the microscope. The pictures of the microstructure of 230
samples where analysed in Leica QWin software, where the amount of non cellular material 231
was measured. In other words, the spaces between cells where analysed. 232
2.8. Texture measurements 233
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Warner-Bratzler shearing blade with a thickness of 3.21 mm, length of 125 mm and width of 234
70 mm was assembled to the TA.XT2® Texture Analyser (Surrey, England). It shears or cuts 235
through the sample with a test speed of 2.5 mm/s. The computer software was set to plot a 236
force versus time plot and the results were expressed as the maximum peak force (shear force 237
in N) required to shear through the sample. Other parameter calculated was the area under the 238
curve which describes the total amount of work required to cut through the sample. This test 239
method incorporates compression of fibres beneath the blade, tension in the adjoining fibres 240
and shearing of the fibres (Bouton, Harris, & Shorthouse, 1975). Textural measurements were 241
performed on samples from the fillet. Each sample was measured three times. 242
2.9. Colour analysis 243
The intensity of the flesh colour was measured by using the MiniScan XE plus from 244
HunterLab using the D65 light source. The instrument records the L* (lightness – intensity of 245
white colour), a* (redness – intensity of red colour) and b* (yellowness – intensity of yellow 246
colour) values. Each sample was measured in triplicates. 247
2.10. Statistical analysis 248
Analysis of variance (ANOVA) was carried out on microbial data and TBARS value in the 249
statistical program NCSS 2000 (NCSS, Utah, USA) to compare the effect of different 250
treatment. The program calculates multiple comparisons using Tukey-Kramer Multiple-251
Comparison Test. The same test was used for the texture and microstructure, using statistical 252
program Sigmastat version 2.03 (Systat Software Inc., California, USA). Results were 253
considered significant at p<0.05. 254
3. Results 255
3.1 Characteristics of cold smoked salmon 256
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The results for dry matter, salt content, fat content and TBARS were presented as mean of 257
two or three values (mean ± SD). The cold smoked salmon contained 37.2 g/100 g ± 0.4 dry 258
matter and 3.2 g NaCl/ 100 g ± 0.1 corresponding to 4.9 % ± 0.2 % water phase salt (WPS). 259
The fat content was 9.6 g/100 g ± 0.4 and pH was 6.1 ± 0.1. The TBARS value was low, e.g. 260
3.9 μmol/kg ± 0.1 μmol/kg. 261
TVC on LH and LAB count on NAP was determined in cold smoked salmon at different 262
processing steps, in the raw filet, after salting and washing and after the smoking step (Fig. 1). 263
The bacterial count increased from <1 log cfu/g to 2.6 log ± 0.2 log cfu/g and to 2.7 log ± 0.1 264
log cfu/g, respectively. The increase of TVC occurred after the salting and washing step while 265
count of LAB increased steadily during the whole process and in the final product LAB were 266
the dominant flora of the total count. It is well known that most of LAB will grow on LH and 267
therefore it is stated that LAB is dominant of the spoilage flora in the samples. Neither 268
Listeria nor Bacillus spores were detected in any of these samples. Neither Listeria nor 269
Bacillus spores were detected in any of these samples. 270
3.2 Challenge test with Listeria innocua 271
The number of L. innocua in overnight culture was 2.03×109 cfu/ml. Cold smoked salmon 272
was spiked with 104 dilution of overnight culture and the final number of L. innocua in the 273
spiked samples was 4.5×103 cfu/g. Increasing the pressure from 400 MPa to 900 MPa had a 274
significant effect on the reduction, from >110 cfu/g to <0.3 cfu/g after 10 s (Table 1). At 400 275
MPa to 500 MPa the reduction of bacteria was less than 1 log to1.5 log cycle. By increasing 276
the pressurization time at 600 and 700 MPa the reduction increased. These results indicate 277
that the pressure needs to be up to 700 MPa to 900 MPa to be efficient enough to reduce the 278
number of L. innocua in cold smoked vacuum packed salmon with regards to the safety of the 279
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product. The initial number of the bacterium in the spiked product tested was rather high, 280
around 4500 cfu/g, which is not the real situation on the market. 281
3.3 Storage trials with spiked samples of cold smoked salmon 282
3.3.1 Microbial analysis 283
The initial bacterial counts in the samples are described in chapter 3.1. No Listeria spp. was 284
detected in unspiked-unpressurised control samples, not even after 41 days storage at 5.5 °C 285
(Fig.2 and Fig.3), indicating a good quality and safety of the raw material used in this 286
experiment. Fig.2 shows that L. innocua was reduced significantly (p< 0.05) after 60 s at 500 287
MPa. One log reduction was observed and the number decreased during storage. No reduction 288
was observed after 10 s and it remained unchanged until after more than 26 days. By 289
increasing the treatment time to 20 and 30 s it appeared that the bacteria were shocked in 290
some way as the total number decreased during the first 12 days of storage but after that the 291
bacteria recovered and the number increased. No L. innocua was detected 5 days after HPP 292
treatment at 900 MPa (Fig. 3) but after storage at 5.5 °C for 26 days to 41 days low level (0.3-293
20 cfu/g) was detected. It is difficult to say if this indicates that some of the cells survived and 294
could recover after some storage time or this difference is due to individual difference 295
between samples. Tables 2 and 3 depicts changes in the TVC and LAB counts in the vacuum- 296
packed cold smoked salmon after pressure treatment and during storage at 5.5 °C. The LAB 297
count was considerably higher than TVC or log 5.5 compared to log 3.7 in untreated samples 298
indicating that by using LH at 15 °C some LAB can not grow (Table 2). Unfortunately the 299
LAB was not identified down to species. At day 5 the TVC count was 0.2-1.2 log lower in 300
samples treated at 500 MPa compared to untreated samples (Table 2). During the storage the 301
number increased steadily and after the 41 days storage about 0.8-2.7 log increases was 302
observed. The number of LAB reduced significantly (p<0.05) compared to untreated samples 303
but during storage 4-6 log increase was observed indicating that the LAB can recover quickly 304
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after HPP treatment at 500 MPa. When the samples were treated at 900 MPa pressure a 305
significant (p<0.05) reduction of TVC of log 2-3 was observed when compared to the non 306
pressure treated samples on day five. The reduction was 2.2 log after 10s, 2.7 log after 20s, 307
1.6 log after 30s and 1.9 log after 60s (Table 3) indicating a better effect when the treatment 308
time was shorter. It should be pointed out that the number increased more rapidly after 309
treatment at 500 MPa time indicating that the cells are more damage after treatment at 900 310
MPa. The effects on LAB count was very clear. After treatment at 900 MPa 2.9 – 4.1 log 311
decrease was observed and after storage for 26 days the count was 0.7-2 log lower after 312
treatment for 30 and 60 s compared to 10 and 20 s indicating better effect after longer treating 313
time. The HPP treatment had significant (p<0.05) effect on the growth of LAB, both at 500 314
and 900 MPa and it was obvious that the recovery after 900 MPa treatments was difficult. A 315
reduction of 4 log were observed after treatment time for 20-60 s at 900 MPa. After storage 316
for 26 days the LAB count was 3.4-6.1 log CFU/g in 500 MPa treated samples compared to 317
8.1 log CFU/g in untreated samples indicating that LAB are sensitive to HPP treatment. The 318
number of LAB on NAP was up to 1.2 – 4 log higher than total count on LH after treatment at 319
500 MPa. The difference was not as high after 900 MPa. The increase of LAB was 320
considerably faster after HPP treatment at 500 MPa (Table 2) indicating that they can recover 321
more quickly maybe because of the lack of competition from background flora. After HPP 322
treatment at 900 MPa LAB was clearly so damaged that they could not recover even after 323
some storage time as shown in table 3. Bacillus spores were not detected in any samples. 324
3.3.2 Lipid oxidation 325
Fig. 4 shows the results from measurements on lipid oxidation. The TBARS was low (4.7 - 326
7.3 μmol/kg). All the smoked fillet samples showed slightly higher TBARS values than the 327
fresh fillet samples (Fig. 4). The difference is not significant (p>0.05). When treated at 900 328
MPa for 60 s, then the TBARS value decreased during storage from 7.31 μmol/kg after 18 329
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days to 5.91 μmol/kg after 41 day. This difference is probably due to the individual difference 330
between samples 331
3.4 Microstructure 332
With increased pressure and longer processing time the space between the myofibrils (cells) 333
increased (Fig. 5). Shrinkage on myofibrils is negligible at 400 and 500 MPa, for processing 334
time of 10 and 20 s. At 600 MPa and higher and for processing time of 30 and 60 s the effects 335
showed a considerable shrinkage of myofibrils resulting in increased space between the 336
myofibrils. 337
3.5 Texture measurements 338
Our results showed non-significant differences in toughness between the control sample and 339
various HPP treatments for 20 s (Fig. 6). The control sample had a large standard deviation 340
and the reason is that the control samples were collected from various part of the fillet, but not 341
from the same location on the fillet. Generally there was a gradual increase in toughness as 342
the pressure was increased. Samples processed at 900 MPa in 10, 30 and 60 s were 343
significantly tougher than samples processed at 400 MPa (data not shown). Each sample was 344
measured in triplicates. Similar effects were also observed for 10, 30 and 60 s processing 345
times, for different HPP treatments. 346
3.6 Colour analysis 347
The effect of high-pressure processing on lightness in the smoked salmon could be seen 348
visually. The lightness increased from the control to 900 MPa when processed for 20 s (Fig. 349
7). The effects were similar for salmon processed for 10, 30 and 60 s (data not shown). The 350
effects are more visible for the interior of the salmon than the surface. There was a significant 351
increase in lightness (L*values) in all samples processed with high pressure treatment 352
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compared to the control (Fig. 8). On the contrary, there was no effect of the high-pressure 353
process on the redness (a* value) of the smoked salmon (Fig. 9). 354
4. Discussion 355
The effects of HPP on micro-organisms in food are of great interest and it has been shown 356
that the effect of the HPP treatment on Listeria monocytogenes. is depending on processing 357
parameters such as time and pressure (Ritz et al., 2000). Investigation of the effect of both 358
pressure and treatment time on the inactivation of L. innocua was conducted. The results 359
indicate that the pressure needs to reach 700-900 MPa to reduce the number of L. innocua to 360
the level where the safety of the product can be ensured and are not detectable (<0.3 cfu/g) 5 361
days after the HPP treatment. HPP above 600 MPa delays the lag phase which can 362
consequently prolong the storage time. However, the initial number of the L. innocua in the 363
final product tested was rather high or 4500 cfu/g which might not reflect the situation in the 364
industry. Beaufort, Rudelle, Gnanou-Besse, Toquin, Kerouanton, Bergis, Salvat and Cornu, 365
(2007) and Cortesi, Sarli, Santoro, Murru and Pepe (1997) stated that typical contamination in 366
naturally contaminated product may be about or below 1 cfu/g . Jørgensen and Huss (1998) 367
reported that initial numbers of L. monocytogenes in cold-smoked salmon were <10 cells/g 368
and only two samples (of 32 positive) contained between 103 and 104 cfu/g after 3-7 wk of 369
storage. In this study Listeria spp. was not detected in naturally contaminated salmon after 370
storage for 41 day at 5.5 °C. The data presented in this paper indicates that Listeria spp. is 371
more sensitive to HPP than the background flora tested. As already explained then the results 372
show that LAB became predominant of the spoilage flora in the samples. It have been stated 373
that LAB often dominates the microbial flora in smoked fish products during refrigerated 374
storage (Magnusson & Traustadottir, 1982). As a result smoked fish products have a 375
prolonged shelf life, since the Gram-negative spoilage flora is somewhat inhibited (Jeppesen 376
& Huss, 1993) and that LAB appear to be well adapted in vacuum packages and more 377
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resistant than Gram negative bacteria to the high salt content found in smoked salmon 378
products (Hansen, Gill & Huss, 1995). The number of LAB on NAP was up to 1.2 – 3 log 379
higher than total count on LH after treatment at 500 MPa. The difference was not as high after 380
900 MPa or mainly below 1 log. The difference observed on the LH and NAP is relative high 381
in the present study compared to the literature (Leroi, Joffraud, Chevalier & Cardinal, 2001). 382
It is expected that LAB can grow on LH. It can be speculated that processing parameters for 383
smoked products such as dry salting and brine injection influence the microbial spoilage 384
(Hansen et al., 1995). In this study the fillets were salted in brine, 8 g NaCl in 100 mL water, 385
for 24 hours, and smoked at 24 hours at 18 to 20 °C. It can also be assumed that not all 386
species of LAB grow well on LH due to the lack of glucose in this medium, or because of the 387
low incubation temperature (15 °C). This difference was also detected in the untreated 388
samples and after five days it was 2 log which is more in accordance with previously reported 389
results (Leroi et al., 2001) but after 41 days it was 4 log. The increase of LAB was 390
considerably faster after HPP treatment at 500 MPa, compared to TVC indicating that they 391
can recover more quickly, possibly because of the lack of competition from other background 392
flora. The results indicate that HPP can extend the microbial shelf-life of cold smoked salmon 393
when exposed to 900 MPa to 26 days compared to suggested limited shelf life for a period (14 394
– 21 days) where growth of Listeria spp. is unlikely to take place or to reach levels >100 cfu/g 395
(Huss et al., 2000). 396
There was no indication of lipid oxidation which is in accordance with previously reported 397
results (Espe, Nortvedt, Lie & Hafsteinsson, 2002; Espe, Nortvedt, Lie & Hafsteinsson, 398
2001). Because of high levels of polyunsaturated lipids the salmon is susceptible to 399
deterioration by oxidation that can affect product quality of the salmon and affect the sensory 400
quality, but values has not been found to be so high that the fish should be regarded as 401
oxidized (Espe et al., 2002). All the values obtained in this study were considered low and 402
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safe for consumption if compared to limits for frozen fish that is 18 μmol/kg according to 403
Robles-Martinez and Cervantes (1982). 404
The effect of HPP on the microstructure of cold smoked salmon was minimal at 400 MPa, but 405
increased with both time and pressure and are most significant at 900 MPa and 60 s. When 406
Fig. 5 is compared to results in Table 1, where the greatest reduction of L. innocua occurs, it 407
is clear that the effect on microstructure coincide with the reduction of the bacteria. The effect 408
at 500 MPa was not as obvious. It is known that HPP affects microstructure of meat and fish 409
at pressure between 200 and 400 MPa, depending on the type (Ledward, 1998). 410
Gudmundsson and Hafsteinsson (2001) have also reported the effect of HPP at 300 MPa on 411
gaping in salmon fillets. Their results indicated more gaping in salmon fillets, caused by 412
shrinkage of the myofibrils and increased space between the myofibrils. 413
The effect of HPP on the textural properties is not clear. It is likely that toughness decreases 414
when salmon fillets were treated at 400 and 500 MPa for 10, 20, 30 and 60 s, compared to the 415
control sample. When pressure was increased to 700 MPa and up to 900 MPa, the toughness 416
was similar as in the control sample. The probable reason for this is that the measurement of 417
the textural properties was performed on samples, located in different parts of the fillets. 418
Jonsson, Sigurgisladottir, Hafsteinsson and Kristbergsson (2001) and Hafsteinsson, Jonsson, 419
Lie, Nortvedt, Thomassen, and Torrissen (1999) reported the difference in toughness on 420
different location in fresh Atlantic salmon fillets. The study of Jonsson et al., (2001) showed 421
variation in the value of maximum force, to cut through the samples along the fillet of fresh 422
salmon from 15 N near the head and 42 N near the tail. Studies have shown increased tissue 423
firmness in fresh bluefish at high pressure (200 MPa for 10 minutes) (Simpson, 1998; Ashie 424
& Simpson, 1996). 425
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Previously it has been shown that the colour of the fish is the main quality aspect of interest to 426
consumers both in the German and the French market (Torrissen et al., 2000; Sigurgisladottir, 427
Torrisen, Lie, Thomassen, & Hafsteinsson, 1997). Negative effects on the colour, caused by 428
HPP of smoked salmon, could therefore be a hindering factor regarding the application of this 429
new technology. The results of this study show no effect on the redness, but Amanatidou et al. 430
(2000) had earlier reported a significant reduction of redness caused by HPP. HPP has 431
immediate effect on the lightness. Even treatment time of 10 s and pressure of 400 MPa had 432
significant effect on the lightness of cold smoked salmon. Lightness then increased as a 433
function of treatment time and pressure and coincides with the visual changes where the 434
salmon became lighter in colour as a function of both time and pressure. However, due to the 435
short processing time the highest value for lightness never exceeded 62. A threshold value of 436
acceptability for smoked salmon has not been reported, but for fresh salmon a threshold value 437
of 70 is considered as unacceptable (Amanatidou et al., 2000). Studies on cod - and mackerel 438
muscle showed that the colour of the muscle became lighter with increasing pressure 439
(Ohshima et al., 1993). Because of the cost of HPP it would be desirable to increase treatment 440
pressure and keep the treatment time as short as possible (Chen et al., 2006). It should be 441
pointed out that the high temperature (42°C) during the holding time (after adiabatic heating) 442
does have an obvious effect on the appearance and on the texture of cold smoked salmon. 443
Therefore it is important to carry out this technique with cooled condition. 444
Conclusion 445
The present study yielded significant results for cold smoked salmon food processing and 446
revealed that the combination of high pressure and short treatment time is very effective to 447
improve the safety of cold smoked products. However further studies are necessary and 448
hurdle effects of some parameters should be evaluated due to the changes in the visual 449
appearance and texture. For example, further studies on the effect of MAP in combination 450
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with HPP should be performed. Decreasing the working temperature during HPP treatment 451
will most likely reduce the effect on microstructure and texture. This new development is 452
promising to meet the requirement for prolonged shelf life of ready-to-eat cold smoked 453
salmon with high microbiological quality and safety. 454
Acknowledgements 455
This work was funded by the Icelandic Research Council. Thanks are extended to Icelandic 456
companies Rifos and Reykofninn and to the Technical University of Berlin (TUBER), 457
Department of Food Biotechnology and Food Process Engineering for assistance with the 458
experiments. Special thanks to Margret Bragadottir, Asa Thorkelsdottir and the staff of 459
microbiological and chemical lab at IFL and IceTech. 460
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Table 1. Survival of L. innocua in HPP treated cold smoked salmon. Most Probable 1
Number (MPN). Samples measured two days after high pressure treatment. Average of 2
two experiments. 3
cfu/g (MPN)
High pressure (MPa) 10 sec 20 sec 30 sec 60 sec
spiked 400 MPa >110 >110 >110 >110
spiked 500 MPa >110 >110 >110 110
spiked 600 MPa >110 16,7 1,9 0,3
spiked 700 MPa 0,8 <0,3 0,3 <0,3
spiked 800 MPa 0,3 <0,3 <0,3 <0,3
spiked 900 MPa <0,3 <0,3 <0,3 <0,3
unspiked 500 MPa <0,3 <0,3
unspiked 900 MPa <0,3 <0,3
4
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Table 2. Microbial evaluation in vacuum packed cold smoked (spiked) after HPP treatment at 500 MPa during storage at 5.5°C. Means of two samples (mean±SD).
Total viable psychrotrophic count on LH (log CFU/g) Lactic acid bacteria on NAP (log CFU/g)
Treatment time (sec)
0 10 20 30 60 0 10 20 30 60
Storage time (days)
5 3,68±1,21 2,86±0,64 2,52±0,04 3,49±0,96 2,80±0,12 5,55±0,77 1,45±0,64 2,45±0,04 2,69±0,56 2,18±0,04
12 3,47±0,38 2,27±0,52 2,48±0,14 na 3,24±0,80 5,40±3,03 3,02±2,01 2,24±0,14 3,58±1,8 3,77±2,81
26 4,71±0,56 3,65±0,04 3,22±0,71 5,28±1,44 4,27±0,18 8,14±0,09 6,10±0,72 3,42±1,43 6,09±1,54 5,38±0,18
41 4,80±0,96 4,60±1,69 5,20±0,12 5,22±0,57 3,56±0,79 8,78±0,24 7,58±0,12 6,44±1,22 7,79±0,91 6,30±0,85 Table 3. Microbial evaluation in vacuum packed cold smoked (spiked) after HPP treatment at 900 MPa during storage at 5.5°C.
Total viable psychrotrophic count on LH (log CFU/g) Lactic acid bacteria on NAP (log CFU/g)
Treatment time (sec)
0 10 20 30 60 0 10 20 30 60
Storage time (days)
5 3,68±1,21 1,45±0,21 1,00±0 2,11±1,57 1,79±1,12 5,55±0,77 na 1,30±0 1,57±0,81 1,00±0
12 3,47±0,38 1,45±0,21 1,30±0,43 1,00 ±0 1,00 ±0 5,40±3,03 4,52±1,01 1,15±0,21 1,39±0,55 1,00±0
26 4,71±0,56 2,29±0,30 2,99±0,27 1,69±0,12 1,45±0,21 8,14±0,09 3,11±0,86 4,00±0,48 2,00±0 2,37±0,52
41 4,80±0,96 na* ng na 1,00±0 8,78±0,24 na na na 1,00±0
* na – no growth detected
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1
2
3
4
Raw fillet Fillet after saltingand washing
Cold smokedsalmon
Log
Num
ber/
g
1
Fig. 1. Distribution of bacteria during processing of cold smoked salmon. Total viable 2
psychrotrophic counts (TVC) determined by spread plating on LH (Long and Hammer ■) 3
and lactic acid bacteria (LAB) on NAP (Nitrite Actidione Polymyxin □). Mean of two 4
samples (mean±SD) 5
6 7
0
1
2
3
4
5
5 d 12 d 26d 41d
Storage time, days
Log
num
ber/g
8
Figure 2. Growth of Listeria innocua (MPN) during storage of cold-smoked salmon at 9
5,5°C. Cold smoked salmon had been treated with 500 MPa for 10 (-□-), 20 (-x-), 30 (-♦-) 10
and 60 (-■-)s. Samples not treated with HPP used as control (unspiked-◊-, spiked-▲-). 11
Mean of two samples (mean±SD). Listeria concentration before pressure was 4.5×103 cfu/g. 12
13
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1
2
3
4
5
5 d 12 d 26d 41d
Storage time, days
Log
num
ber/
g
14 Figure 3. Growth of Listeria innocua (MPN) during storage of cold-smoked salmon at 15
5,5°C. Cold smoked salmon had been treated with 900 MPa for for 10 (-□-), 20 (-x-), 30 (-♦-) 16
and 60 (-■-)s. Samples not treated with HPP used as control (unspiked-◊-, spiked-▲-). 17
Mean of two samples (mean±SD). Listeria concentration before pressure was 4.5×103 cfu/g. 18
19
0
2
4
6
8
10
12
Fresh sample
Control 500 MPa-10s
700 MPa-10s
900 MPa-10s
500 MPa-30s
700 MPa-30s
900 MPa-30s
500 MPa-60s
700 MPa-60s
900 MPa-60s
TBA
(µm
ol/k
g)
20
Fig. 4. Changes in TBARS-value as an indicator for rancidness during storage at 5.5°C for 21
18 (black column) and 41 (blank column) days after HPP treatment at 500, 700 and 900 22
MPa for 10, 30 and 60s Mean of two samples (mean±SD) 23
24
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Control
400 MPa
500 MPa
600 Mpa
700 Mpa
800 Mpa
900 MPa
10 sec 20 sec 30 sec 60 sec 1
Fig. 5. Effect of high pressure on the microstucture of cold smoked salmon. Frozen 2
specimen were prepared two days after treatment and stored at -80°C until examined. 3
4
MANUSCRIP
T
ACCEPTED
ARTICLE IN PRESS
1
0
5
10
15
20
25
30
0 400 500 600 700 800 900
Pressure (MPa)
Toug
hnes
s (N
)
1
Fig. 6. Effect of high pressure process for 20 s on smoked salmon. Samples measured two 2
days after high pressure treatment. Mean of three samples (mean±SD). 3
4
5
Fig. 7. Photographs of high-pressure processed smoked salmon for 20 s. Photos taken two 6
days after high pressure treatment. 7
8
Co
Co
ntrol
ntrol
400 MPa
400MPa
500 MPa
500MPa
600 MPa
600MPa
700 MPa
800 MPa 900 MPa
MANUSCRIP
T
ACCEPTED
ARTICLE IN PRESS
2
0
10
20
30
40
50
60
70
0 400 500 600 700 800 900
Pressure (MPa)
L va
lue
9 Fig. 8. The lightness (L) of the high-pressure processed smoked salmon (20 sec) measured by 10
MiniScan from HunterLab. Samples measured two days after high pressure treatment. Mean of 11
three samples. (mean±SD). 12
13
0
5
10
15
20
25
30
35
0 400 500 600 700 800 900
Pressure (MPa)
a va
lue
14 15 Fig. 9. The redness (a) of the high-pressure processed (20 sec) smoked salmon measured by MiniScan 16
from HunterLab. Samples measured two days after high pressure treatment. Mean of three. 17
(mean±SD). 18
19
