Fermentation of salmon fillets with a variety of lactic acid bacteria

Food Research International, Vol. 30, No. 10, pp. 777±785, 1997 # 1998 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0963-9969(98)00045-3 0963-9969/98/$19.00+0.00

Fermentation of salmon ®llets with a variety of lactic acid bacteria Martine Morzel,ab Gerald F. Fitzgeraldc & Elke K. Arendta* a Department of Food Technology, University College Cork, Western Road, Cork City, Ireland National Food Biotechnology Centre, University College Cork, Western Road, Cork City, Ireland c Department of Microbiology, University College Cork, Western Road, Cork City, Ireland

b

The in¯uence of ®ve strains of lactic acid bacteria (four Lactobacillus and one Carnobacterium) on the quality of fermented salmon ®llets was studied. Best starter growth (increase of more than 1 log in 3 days) and acidi®cation of muscle (e.g. pH reduction of approximately 0.7 units in 5 days) were achieved with the two commercial strains L. sake LAD and L. alimentarius BJ33. pH reduction was consistently lower (e.g. reduction of 0.2 units in 5 days) with C. piscicola 85. Protein breakdown as observed on SDS-PAGE gels was similar for all strains. In contrast, the starter strain did in¯uence texture and colour changes. Fast acidifying strains L. sake LAD and L. alimentarius BJ33 brought about a ®rmer overall texture and a lighter colour, while softening of ¯esh occurred in samples processed with C. piscicola 85. Sensory evaluations indicated that samples processed with fast acidifying strains were preferred. L. sake LAD and L. alimentarius BJ33 are regarded as suitable starters for fermentation of salmon ®llets. # 1998 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved Keywords: salmon ®llets, Salmo salar, fermentation, lactobacilli, Carnobacterium piscicola.

INTRODUCTION

enhuÈskes, 1993). Furthermore, bacteriocins produced by certain strains of lactic acid bacteria may o€er additional protection against undesirable bacterial contaminations (Marrug, 1991). Interest in the use of lactic acid bacteria for ®sh products has increased in the past years. Studies have been performed to assess their performance as protective cultures (Jeppesen and Huss, 1993; Leroi et al., 1996) or as starter cultures for processing fermented ®sh sausages or ®sh-meat sausages (Hwang et al., 1989; Aryanta et al., 1991). Production of non-comminuted fermented products has been very limited in the past, although it was shown by Morzel et al. (1997) that whole salmon ®llets can be fermented using a commercially available starter of Lactobacillus sake LAD. The objective of this study was to evaluate the performance of ®ve strains of lactic acid bacteria, including two bacteriocin producers, in the production of fermented salmon ®llets. The in¯uence of individual strains on the product's characteristics during processing and storage was evaluated.

In many European countries, fatty ®shes (salmon, herring, mackerel) are frequently preserved by salting or smoking. In Scandinavia, where such seafood is abundant, spontaneous fermentation has also been exploited in order to extend the shelf-life of fresh ®sh and to induce unique organoleptic qualities (Knochel, 1983). The traditional process involves curing salmon ®llets with a sugar±salt mixture and usually herbs or spices, resulting in so-called gravad ®sh. A very complex ¯ora develops on those products. For example, Leisner et al. (1994) isolated 58 strains of lactobacilli and carnobacteria from gravad salmon. Use of starter cultures allows better control of the ®nal ¯ora of a fermented product and reduces the growth of spoilage and pathogenic microorganisms (Erichsen, 1983; Buck*To whom correspondence should be addressed. Fax: +35321-276318; e-mail: [email protected] 777

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MATERIALS AND METHODS Bacterial cultures Five starter bacteria were used in this study. Lyophilised samples of Lactobacillus sake LAD were provided by GewuÈrzmuÈller (Stuttgart, Germany). Lactobacillus alimentarius BJ33 and Lactobacillus pentosus 03A were supplied by Christian Hansen's laboratory (Hùrsholm, Denmark). The two bacteriocin-producers Lactobacillus sake 38 and Carnobacteria piscicola 85 were obtained from the collection of the Department of Microbiology, University College Cork. With the exception of the strain LAD, which was used directly in its lyophilised form, all strains were prepared according to the following procedure. Strains were grown at 30 C in 10 ml MRS broth (Oxoid, Basingstoke, Hampshire, England) until the stationary phase was attained, i.e. 12±36 h depending on the strain. This was used to inoculate 500 ml MRS broth which was incubated at 30 C overnight. Resulting suspensions were centrifuged in sterile containers at a speed of 10 000 rpm for 10 min and the supernatant was discarded. The pellet was resuspended in approximately 25±30 ml of a sterile 30% (w/w) sucrose solution. This material (5±10 ml) was poured into sterile Petri dishes and placed in a freeze-dryer (Unitop 800L+Freezemobile 12LS, Virtis, Gardiner, NY, USA). Complete drying was achieved after 4±7 days. Dried cultures were ground using a domestic mixer (Krups 708, Limerick, Ireland) which was swabbed with 90% ethanol before each usage. Bacteria were enumerated in the powder by the spread plate technique, on MRS agar (Oxoid, Basingstoke, England) for the lactobacilli and on Elliker agar made of Elliker broth (Difco, Detroit, IL, USA) containing agar (15 g lÿ1) for the C. piscicola 85. Minimal counts of 109 CFU gÿ1 were required. The ®nal sucrose concentration was measured by UV spectrophotometry at a wavelength of 340 nm using an enzymatic kit (Boehringer Mannheim GmbH, Mannheim, Germany). Processing of ®sh ®llets Processing and sampling was carried out under stringent hygienic conditions in order to minimize contamination of the ®llets by environmental microorganisms. Fresh whole skinned salmon ®llets (Salmo salar) were trimmed of visible lateral fat, weighed and stored at 4 C until required for experimental use on the same day. Ten grams (4 g for L. sake LAD) of freeze-dried culture per kg of salmon were weighed and the corresponding weight of sucrose was calculated. Freeze-dried material was supplemented with sucrose and NaCl, in order to reach the ®nal concentration of 10 and 25 g per kg of salmon, respectively. This mixture was applied evenly by rubbing onto the visceral side of the ®llets. Fillets

were then placed on a metal rack and stored at 12 C overnight, to allow the di€usion of the ingredients into the ¯esh. Subsequently, the undissolved ingredients remaining on the surface of the ®llets were removed by scraping them o€ with a knife. Each ®llet was divided into two or three portions which were individually vacuum-packed in polyamide±polethylene bags (Grace Muli¯ex GMBH, Flensburg, Germany). For the texture measurement experiments, whole ®llets were vacuumpacked in the same material. Samples were incubated at 12 C for 72 h and subsequently stored at 4 C for a further 18 days. Sampling Four batches of ®ve ®llets were produced. Two batches were used for monitoring pH, colour and microbiological counts on the raw material, after storage overnight at 12 C (day 0) and on days 1, 2, 3, 5, 11, 16 and 21 of processing. For these analyses, one pack of each treatment was randomly chosen on each sampling day. Another batch was used for rheological tests, which were performed on the raw material and on days 0, 3, 12 and 21. Samples for gel electrophoresis were taken from the same batch and it was performed on raw material and on days 7, 14 and 21. Finally, another batch of ®ve ®llets was used for sensory evaluation, which took place on days 5 and 11 of processing. Enumeration of bacteria in salmon ®llets Ten grams of salmon were aseptically sampled from one vacuum-packed portion and homogenised for 2 min at high speed in 90 ml of Ringers solution (Oxoid) using a Stomacher 400 (Seward Medical, London, UK). The homogenate was serially diluted in Ringers solution and used for enumeration of microorganisms. Lactobacilli were enumerated on MRS agar, while carnobacteria were enumerated on Elliker agar made as described previously. pH and colour measurements pH was taken in triplicate from one vacuum-packed portion directly in the muscle, using a spear probe pH meter PH62 (WTW, Weilheim, Germany). Colour was monitored using a Chroma-Meter CR300 (Minolta, Osaka, Japan). Five readings were taken of each sample vacuum-packed in transparent plastic bags and the average value was calculated. Chromatic values are given in the CIE L*a*b* system (Anon., 1991). Sensory evaluation Samples were evaluated by 15 untrained panelists, recruited on the single criterion of their liking for fresh

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779

and smoked salmon. On the day of the evaluation, thin slices were cut from the central part of each ®llet (i.e. avoiding head and tail), wrapped individually in tin foil, labelled with a three-digit code and stored at 4 C until required. Panelists were asked to rate each sample on a 9-point hedonic scale (1: dislike extremely, 5: neither like nor dislike, 9: like extremely). Crackers and water were provided and assessors were asked to cleanse their palate between each sample. Written description of the products was also encouraged. Statistical analysis (analysis of variance and paired comparisons using the repeated measures design) was carried out using SPSS 6.1. (SPSS Inc., Chicago, IL, USA). Texture measurements Texture measurements were performed with a Texture Analyser TA-XT2i (SMS, Goldaming, UK) equipped with a load cell of 25 N. Muscle cores with a diameter of 35 mm and a height of 12±15 mm were sampled from salmon ®llets, taking care to avoid the ®brous area surrounding the central axis. Samples were placed on the TA base with the inoculated surface facing up. A 6 mm diameter stainless steel cylinder was used to carry out a penetration test. For each sample (speci®c day, speci®c strain) 8 measurements were performed on a minimum of two di€erent cores. The probe was moved to the surface of the sample and it proceeded to penetrate the ¯esh to a depth of 8 mm. Force was plotted versus distance of travel of the probe. Examples of resulting curves are presented in Fig. 1. They showed either several peaks (thick line on the graph) corresponding to muscles ruptures, or a maximum value at the end of the probe travel (®ne line on the graph) when no irreversible breakdown of the muscle structure occurred. Data recorded for analysis were d (mm)=rupture distance, i.e. distance at ®rst peak; F (N)=force at ®rst peak and A (N mmÿ2)=work (area under the curve) at ®rst peak. When no peak was observed after 8 mm, d was recorded as 8, F was equal to the ®nal strain, and A was the total area under the curve. SDS-PAGE gel electrophoresis SDS-PAGE was performed by the method of Laemmli (1970), using 10% polyacrylamide gels in a Mini-protean dual slab cell (BioRad, Richmond, USA). Samples were prepared as follows: 5 g of muscle (free of brown ¯esh) were homogenised with 20 ml of distilled water with an Ultra-Turrax (Janke & Kunkel GmbH, Staufen, Germany) set at a speed of 13 500 rpm for approximately 10 s. The suspension was poured into Petri dishes and freeze-dried overnight. Lyophilised samples (20 mg corresponding to approximately 15±16 mg protein) were then placed in 3 ml of SDS sample bu€er, mixed using an agitator (Minishaker MS1, Janke & Kunkel GmbH, Staufen, Germany) for 10 s and boiled for

Fig. 1. Typical curves obtained in a penetration test exhibiting muscle rupture (thick line) or not (®ne line).

1 min. Aliquots of 0.5 ml were frozen. Prior to use, samples were thawed by placing in boiling water for 1 min and 5 l of this material was loaded per well. RESULTS AND DISCUSSION Microbiological analyses Counts on MRS or Elliker agar were considered to be counts of the starter cultures: only one type of colonies (colour, size) were present on the plates, suggesting that contamination from the environment, if any, was very limited. Initial levels of starter cultures in the salmon samples varied from 1.8107 to 3.7108 CFU gÿ1. Bacterial counts achieved by day 3, 5 and 16 and incremental growth values (=
log CFU gÿ1) between sampling days are presented in Table 1, for the two independent experiments. For the lactobacilli, while absolute values di€ered from one trial to another, the trends were similar in both experiments. Counts of the `control' strain L. sake LAD increased rapidly at 12 C and attained their maximum value on day 3. Subsequent storage at 4 C resulted in a decrease in the counts, as measured on days 5

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Table 1. Bacterial counts of di€erent starter cultures (CFU gÿ1) on days 0, 3, 5, and 16 of processing and incremental growth (=
log CFU gÿ1) between two sampling days, in two independent experiments Day

L. sake LAD

L. alimentarius BJ33

L. pentosus 03A

Counts

1.3108/7.5107

2.6107/7.8107

1.1108/2.3 108

1108/1.8107

3.7108/4.5107

Counts Growth

1.8109/2.9109 1.14/1.59

1.1109/1.9109 1.63/1.39

2.6108/7.5 107 0.37/ÿ0.49

1109/3.2108 1/1.25

6.1108/1.6108 0.22/0.55

Counts Growth 16 Counts Growth

1.4109/1.4109 ÿ0.11/ÿ0.22

1.1109/2.3109 0/0.08

1.7108/2.7 108 ÿ0.17/0.56

4.8108/1.5108 ÿ0.32/ÿ0.33

7.5108/5.4108 0.09/0.53

6.1108/1.1109 ÿ0.36/ÿ0.10

1.5109/1.6109 0.13/ÿ0.16

1.1109/9.5 108 0.8/0.55

6108/5.1108 0.09/0.53

1.4109/9.5108 0.27/0.14

0 3 5

and 16. Growth of the bacteriocin producing L. sake 38 was slightly lower during the fermentation period (day 3 results) than in the commercial L. sake LAD samples. A decrease in the counts was observed by day 5, but a second growth phase occurred at 4 C. L. alimentarius BJ33 showed the best growth at 12 C (day 3 results) in both trials and counts remained approximately constant over the storage period at 4 C. The psychrotrophic character of this strain has previously been reported in the literature: L. alimentarius is found in marinated ®sh (Stiles, 1996), usually kept at 4±8 C. In contrast, the population of L. pentosus 03A decreased or showed very limited growth at 12 C. Further growth was slow to develop. Counts of C. piscicola 85 di€ered largely in the two trials. Nevertheless, growth in the ®rst three days was limited (<1 log) in both experiments, and counts

L. sake 38

C. piscicola 85

subsequently increased steadily at 4 C. This agrees with Mauguin and Novel (1994) who stated that C. piscicola NCDO 2762 was able to grow in a ®sh model system at 5 C unlike 5 Lactobacillus strains examined. Physical characteristics pH The pH values of the samples observed over the 21 day duration of the second trial are presented in Fig. 2. pH drop during the fermentation phase of processing was approximately similar for all strains (0.4±0.6 units). However, clear di€erences could be observed during storage at 4 C. The two strains L. sake LAD and L. alimentarius BJ33 induced a rapid pH drop in salmon ®llets, especially from day 3 to 11 of processing. A small

Fig. 2. pH drop observed in salmon ®llets processed with ®ve di€erent strains of lactic acid bacteria, from raw material (day ÿ1) to end of storage (day 21). Each point represents the average of three values.

Fermentation of salmon ®llets with a variety of lactic acid bacteria pH increase occurred in both cases after day 11. A pH increase was also observed in pork sausages manufactured with a mixed starter culture of micrococci/lactobacilli after 7 days (Soultos et al., 1992) and generally in fermented sausages in later stages of the ripening period (Roca and Incze, 1990). pH generally decreased less rapidly in samples processed with L. pentosus 03A, probably correlating with poor bacterial growth. Acidi®cation was even slower in samples processed with L. sake 38 and C. piscicola 85. In the ®rst trial, di€erences between strains were less pronounced, which may be explained by the overall lower bacterial growth. However, L. sake LAD and C. piscicola 85 were again the fastest and slowest acidi®ers, respectively. The weak ability of C. piscicola to reduce pH in a ®sh system was also reported by Mauguin and Novel (1994). Colour All colour coordinates followed the same evolution during processing, i.e. they decreased during storage overnight at 12 C, before increasing throughout the remaining period of the trial. Values, a* and b* (results not shown) initially decreased by 3±8 and 4±10 units, respectively, indicating a loss of redness and yellowness compared to fresh salmon. No consistent di€erence was observed between samples, as only a limited fermentation could have occured after 12±15 h. It can be assumed that the loss of redness and yellowness was due to the salting process. During the fermentation and storage period, all samples regained redness and yellowness compared to day 0. Final redness coordinates were 1.8± 3.9 units lower than in fresh salmon, while yellowness

781

coordinates were from 4.7 units lower to 2.8 units higher than in fresh salmon. No relationship between the type of starter and changes in the a* and b* values was observed. In contrast, the strain used for processing did in¯uence the brightness (L* value) of the samples. The results of the second trial are presented in Fig. 3. A decrease of 8±10 units occurred in the ®rst phase of processing (storage overnight), indicating that samples acquired a darker shade compared to fresh salmon, again probably due to salting. All samples increased in brightness to a very similar extent during fermentation at 12 C. However, di€erences between strains were particularly clear from day 5. The 3 strains L. sake LAD, L. alimentarius BJ33 and L. pentosus 03A induced a major increase in brightness (+17.8±21.4 units from day 0 to 21), as opposed to L. sake 38 and C. piscicola 85 where the increase was limited to 10 and 11 units, respectively. A direct relationship between pH on L* values of samples is suggested by the results. Linear regression performed on data from day 0 to 21 showed correlation coecients varying from 0.87 to 0.96 for Lactobacillus strains. The relationship was not linear in samples processed with the Carnobacterium strain (r2=0.41), probably because there was almost no changes in pH and L* values during the experiments. In the ®rst trial, very little di€erence was detected between the three strains L. sake 38, L. alimentarius BJ33 and L. pentosus 03A, as observed for the pH. The L. sake LAD samples had also the highest L* values and lowest pH. The hypothesis regarding the in¯uence of pH on brightness of samples processed with lactobacilli strains is maintained.

Fig. 3. L* value observed in salmon ®llets processed with ®ve di€erent strains of lactic acid bacteria, from raw material (day ÿ1) to end of storage (day 21). Each point represents the average of ®ve values.

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Texture Three parameters (rupture distance d, force F and work A at ®rst peak) were recorded for texture studies. Force varied between 3 and 5 N for all strains and all days but there was no apparent di€erence between any of the samples (results not shown). In contrast, distance (d) and work (A) followed de®nite patterns as shown in Figs 4 and 5, respectively. Work values of shear or penetration tests have been used to assess toughness of meat products (Szczesniak, 1966; Bourne, 1978). d Values are indicators of the resistance to deformation. They increase with the overall softness and pliability of a material, or when its surface is strengthened. Penetration tests using either a 10 mm cylinder or 1 mm diameter needle have been carried out on cooked ®sh (Johnson et al., 1981; Hatae et al., 1984). Conclusions of the work suggested that it was possible to di€erentiate textures of de®ned species. However, textural properties of cooked ®sh are by no mean comparable to raw ®sh, as thermal degradation of collagen brings about a total loss of binding properties of the connective tissue. In fermented products, where the maximum temperature during processing is 12 C, main parameters that may in¯uence the texture are salt concentration, pH and to a lesser extent the integrity of the myo®brillar proteins (Dunajski, 1979). In the ®rst stage of processing, pH did not change and salt was consequently the main factor in¯uencing the texture. The same salt concentration was used for the production of all samples and texture followed initially a similar trend. Both work and distance increased, indicating a general increase in toughness and a strengthening of the surface in all samples. This agrees with

Sikorski and Ruiter (1994) who described the e€ect of salting on ®sh muscle as bringing about protein denaturation, coagulation and decreased extractibility of the muscle proteins, and general toughening of the ¯esh. In fermented sausages, swelling of myo®brils is also due to salting in early phases of ripening (Katsaras and Budras, 1992) and contributes to the ®rmness of the product. During fermentation and storage of fermented salmon, some of the di€erences in texture parameters are explained partially by the pH of the samples. In poultry and ®sh for example, when the pH approaches the isoelectric point of the myo®brillar proteins, a tightening of the protein structure occurs (Dunajski, 1979; Stewart et al., 1984). This explains the rapid and large work increase for the two fast-acidifying strains L. sake LAD and L. alimentarius BJ33. The increase was pronounced for the L. pentosus 03A samples only after day 3, as was observed also for the pH values. In samples processed with C. piscicola 85, where pH drop was the slowest to occur, work values decreased from day 0. Texture deterioration was favoured because samples did not bene®t from the `protective' e€ect of low pH. However, the mechanism of muscle softening is unclear. A pH of approximately 3.5 and a temperature of 45 C were found by Geist and Crawford (1974) to be the optimal conditions for the activity of sole intramuscular autolytic enzymes (cathepsins). Alkaline proteases with an optimal temperature of 70 C were also reported in salmon muscles (Stoknes and Rustad, 1995). Both studies suggest that these particular enzymes had a very limited acitivity at conditions prevailing in fermented salmon. However, other endogeneous enzymes such as acid

Fig. 4. Rupture distance (mm) as in¯uenced by time and strain used for processing, in a penetration test. Each value is the average of eight measurements.

Fermentation of salmon ®llets with a variety of lactic acid bacteria

783

Fig. 5. Work at ®rst peak (N mmÿ2) as in¯uenced by time and strain used for processing, in a penetration test. Each value is the average of eight measurements.

proteases active at low temperature or lipolytic enzymes may be responsible for texture changes. Microbial enzymes speci®c to C. piscicola 85 could also be implicated. Distance to failure allowed less di€erentiation between samples, possibly because surface characteristics are more a€ected by salting. Nevertheless, increase (surface strengthening) was lower for C. piscicola 85 and late to occur for L. pentosus 03A when compared to the two strains L. sake LAD and L. alimentarius BJ33, con®rming stress observations. Measurements related to L. sake 38 samples showed no trend. SDS-PAGE gel electrophoresis The e€ect of processing with L. alimentarius BJ33 on native proteins of salmon is illustrated in Fig. 6. Other samples showed similar electrophoretic patterns. Proteins of fresh salmon (A) are compared to proteins of the fermented product on days 7 (B), 14 (C) and 21 (D). The majority of protein degradation occurred between days 0 and 7. Contractile proteins, indicated by arrows on the graph, were greatly a€ected. Thus, a decrease in the myosin heavy chain (205 kDa) was observed, probably linked with the appearance of a 190 kDa protein and the accumulation of the pre-existing 155 kDa protein. Actin (42 kDa) was also a€ected. Other changes included the decrease or disappearance of components with molecular weights of 95, 73, 35, 32 and 20 kDa and the appearance of a 78 kDa protein. Degradation of the contractile proteins does not occur in meat products during storage at refrigerated temperatures (Koohmaraie, 1994). In contrast, degradation of the myosin

associated with accumulation of peptides in heated seafood is responsible for the so-called `modori' phenomenon, i.e. a weakening of the gel structure of surimi (Sikorski and Sun Pan, 1994). In the present study, all samples showed similar patterns of protein breakdown, as opposed to texture parameters. This indicates that protein degradation and salt had no e€ect on the textural di€erences between samples. The electrophoretic pattern was not modi®ed even for samples processed with L. pentosus 03A, which was found to be proteolytic on meat (Fransen et al., 1997). Consequently, the protein breakdown appeared to be mainly caused the activity of endogeneous enzymes and not bacterial enzymes. The presence of bacterial proteases speci®c to C. piscicola 85, suggested earlier in the article, was not con®rmed by the electrophoretic pattern.

Fig. 6. Electrophoretic pattern of salmon samples processed with L. alimentarius BJ33 on 10% SDS-PAGE gels. Aliquots of 5 ml (0.005% protein) were applied to each well.

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Table 2. Average sensory scores (x ‹ SD) on days 5 and 11, and e€ect of time, strain and interaction time by strain. Evaluation was carried out by a panel of 15 subjects LAD Odour D5 5.7‹2.5 D11 6.5‹1.6 Flavour D5 7.9‹1 D11 6.7‹2 Texture D5 7.3‹1.4 D11 6.1‹2.3

Sensory scores (days 5±11) BJ33 03A 38

85

Time e€ecta

Strain e€ecta

Interaction time by straina

6.2‹2 6.3‹1.7

5.4‹2.3 4.6‹2

4.8‹2.2 5.9‹2

5.7‹1.9 4.3‹1.9

n.s.

**

*

7.1‹1.7 6.3‹2

6.8‹1.5 6.5‹2

5.7‹2.3 6.5‹2

6.9‹1.6 5.5‹2.2

*

*

n.s.

6.8‹1.6 6.3‹1.8

7.3‹1.4 6.3‹2.3

6.3‹1.9 5.5‹2.6

6.3‹1.9 5.5‹1.7

**

*

n.s.

a n.s. Not signi®cant. *Signi®cant at a 5% level (p0.05); **signi®cant at a 1% level (p0.01).

Sensory evaluation Sensory evaluations were performed on all samples on days 5 and 11 of processing. Table 2 shows average scores and standard deviations, and the e€ect of time, strain and interaction time by strain, respectively. Paired comparisons were performed and p-values below 0.01 were considered signi®cant. Overall, time did not a€ect signi®cantly the `odour' results. In contrast, the strain used and the interaction time by strain had signi®cant e€ects on this attribute. This indicates that di€erences in scores between strains was a€ected by time. As a matter of fact, samples processed with L. sake LAD and L. sake 38 received higher scores on day 11, in contrast with L. pentosus 03A and C. piscicola 85 samples (decreasing scores) and L. alimentarius BJ33 samples (approximately constant scores). On day 11, the L. sake LAD and L. alimentarius BJ33 samples were signi®cantly preferred to the L. pentosus 03A and C. piscicola 85 samples. Odour of products processed with the three strains L. pentosus 03A, L. sake 38 and C. piscicola 85 was scored below the acceptability limit (5) on at least one of the evaluation days. Best overall odour acceptance (de®ned as the average score of the two di€erent days) was exhibited with the two strains LAD and BJ33. Flavour and texture were signi®cantly a€ected by the storage time and strain used. The interaction was nonsigni®cant, i.e. the strain e€ect was not majorly modi®ed by the day of tasting. Flavour acceptance decreased for all samples from day 5 to 11 except for the L. sake 38 samples. Late acidi®cation of muscle may be partially responsible for this result. Three panelists commented on a `raw' ¯avour of this sample on day 5. Furthermore, pH (5.99) was very close to its initial value in fresh salmon. Averaged scores over the two days indicated that the L. sake LAD samples were signi®cantly preferred to the L. sake 38 and C. piscicola 85 samples. Seven panelists described the ¯avour of the L. sake LAD samples as `sour' or `acidic' on day 5.

Texture scores decreased for all strains from day 5 to 11. No statistical di€erence was observed from the averaged scores over the two days. However, comments indicated that the L. sake 38 and C. piscicola 85 samples (scored lower on both days) were perceived as `mushy', `jelly-like' or `soft'. This agrees with the texture observations, as ®sh fermented with C. piscicola 85 decreased in ®rmness as shown by work values. Quality parameters being very inconsistent for L. sake 38, it is assumed that texture deterioration also occurred for this particular batch, due to the slow pH drop. Sensory evaluations highlighted the importance of pH and its direct implications on ¯avour and texture of fermented salmon. Furthermore, the odour of samples was the most discriminative attribute. Fermentation with three strains led to scores below the acceptability limit on at least one of the days of tasting. They should therefore not be used as starter cultures in this speci®c product. L. sake LAD and L. alimentarius BJ33 both produced fermented salmon with very acceptable odour, texture and ¯avour. Further sensory investigations on these speci®c samples would be necessary for the exact determination of the shelf-life of fermented salmon. Based on the sensory data described in this study, the shelf-life can be estimated to approximately 10 days. REFERENCES Anon. (1991) Minolta Chroma Meter CR 300 Instruction Manual. Minolta Co. Ltd, Osaka. Aryanta, R. W., Fleet, G. H. and Buckle, K. A. (1991) The occurrence and growth of microorganisms during the fermentation of ®sh sausage. Int. J. Food Microbiol. 13, 143± 156. Bourne, M. C. (1978) Texture pro®le analysis. Food Technol. 32(7), 62±66. BuckenhuÈskes, H. J. (1993) Selection criteria for lactic acid bacteria to be used as starter cultures for various food commodities. FEMS Microbiol. Rev. 12, 253±272. Dunajski, E. (1979) Texture of ®sh muscle. J. Text. Stud. 10, 301±318.

Fermentation of salmon ®llets with a variety of lactic acid bacteria Erichsen, I. (1983) Fermented ®sh and meat products: present position and future possibilities. In Food Microbiology: Advances and Prospects, ed T. A. Roberts and F. A. Skinners, pp. 271±284. Academic Press, London. Fransen, N. G., O'Connell, M. B. and Arendt, E. K. (1997) A modi®ed agar medium for the screening of proteolytic activity of starter meat cultures for meat fermentation purposes. Int. J. Food Microbiol. 36, 235±239. Geist, G. M. and Crawford, L. (1974) Muscle cathepsins in three species of Paci®c sole. J. Food Sci. 39, 548±551. Hatae, K., Yoshimatsu, F. and Matsumoto, J. J. (1984) Discriminative characterization of di€erent texture pro®les of various cooked ®sh muscles. J. Food Sci. 49, 721±726. Hwang, J. W., Angel, S., Kinsman, D. M. and Hall, K. N. (1989) Preparation of fermented sausages from underutilized ®sh and meat sources. J. Food Process. Preserv. 13(3), 187±200. Jeppesen, V. F. and Huss, H. H. (1993) Antogonistic activity of two strains of lactic acid bacteria against Listeria monocytogenes and Yersinia enterocolitica in a ®sh model system at 5 C. Int. J. Food Microbiol. 19, 179±186. Johnson, E. A., Peleg, M. and Sawyer, F. M. (1981) Mechanical methods of measuring textural characteristics of ®sh ¯esh. Refrig. Sci. Technol. 4, 93±103. Katsaras, K. and Budras, K-D. (1992) Microstructure of fermented sausage. Meat Science 31, 121±124. Knochel, S. (1983) Fermented ®sh in Scandinavia. Kor. J. Appl. Microbiol. Bioeng. 11(4), 347±351. Koohmaraie, M. (1994) Muscles proteinases and meat aging. Meat Sci. 36, 93±104. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680±685. Leisner, J. J., Millan, J. C., Huss, H. H. and Larsen, L. M. (1994) Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted ®sh. J. Appl. Bacteriol. 76, 417±423. Leroi, F., Arbey, N., Jo€raud, J-J. and Chevalier, F. (1996) E€ect of inoculation with lactic acid bacteria on extending

785

the shelf-life of vacuum-packed cold smoked salmon. Int. J. Food Sci. Technol. 31, 497±504. Marrug, J. D. (1991) Bacteriocins, their role in developing natural products. Food Biotechnol. 5(3), 305±312. Mauguin, S. and Novel, G. (1994) Characterization of lactic acid bacteria isolated from seafood. J. Appl. Bacteriol. 76, 616±625. Morzel, M., Fransen, N. G. and Arendt, E. K. (1997) De®ned starter cultures used for fermentation of salmon ®llets. J. Food Sci. 62(6), 1214±1217, 1230. Roca, M. and Incze, K. (1990) Fermented sausages. Food Rev. Int. 6(1), 91±118. Szczesniak, A. S. (1966) Texture measurements. Food Technol. 20(10), 52±58. Sikorski, Z. E. and Ruiter, A. (1994) Changes in proteins and nonprotein nitrogen compounds in cured, fermented, and dried seafoods. In Seafood Proteins, eds Z. E. Zikorski, B. Sun Pan and F. Shahidi, pp. 113±126. Chapman and Hall, New York. Sikorski, Z. E. and Sun Pan, B. (1994) The e€ect of heatinduced changes in nitrogenous constituents on the properties of seafoods. In Seafood Proteins, eds Z. E. Zikorski, B. Sun Pan and F. Shahidi, pp. 84±98. Chapman and Hall, New York. Soultos, N., Vareltzis, K. and Georgakis, S. (1992) Lefkasstyle fermented sausages manufactured by adding di€erent starter cultures. Fleischerei 43(8), 3±6. Stewart, M. K., Fletcher, D. L., Hamm, D. and Thomson, J. E. (1984) The in¯uence of hot boning broiler breast muscle on pH decline and toughening. Meat Science 63, 1935±1939. Stiles, M. E. (1996) Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek 70, 331±345. Stoknes, I. and Rustad, T. (1995) Proteolytic activity in muscle from Atlantic salmon (Salmo salar). J. Food Sci. 60(4), 711± 714.

(Received 16 October 1997; accepted 13 June 1998)