Microbiological, biochemical and sensory assessment of mussels (Mytilus galloprovincialis) stored under modified atmosphere packaging

Journal of Applied Microbiology 2005, 98, 752 760 doi:10.1111/j.1365-2672.2004.02512.x Microbiological, biochemical and sensory assessment of mussels (Mytilus galloprovincialis) stored under modified atmosphere packaging A.E. Goulas, I. Chouliara, E. Nessi, M.G. Kontominas and I.N. Savvaidis Laboratory of Food Chemistry and Microbiology, Department of Chemistry, University of Ioannina, Ioannina, Greece 2004/0545: received 13 May 2004, revised 4 August 2004 and accepted 7 October 2004 ABSTRACT A.E. G O U L A S, I. C H O U LIARA, E. N E S S I, M. G. K O N T O M I N A S A N D I.N. S A V V A I D I S. 2004. Aims: To determine the microbiological, biochemical and sensory changes of mussels during storage under aerobic, vacuum packaging and modified atmosphere packaging (MAP) conditions at 4 C, and to determine shelf-life of mussels under the same packaging conditions using the above assessment parameters. Methods and Results: Aqua-cultured mussels (Mytilus galloprovincialis) were obtained from a local culture farm, packaged aerobically under VP and MAP (50%/50% : M1, 80%/20% : M2, 40%/30%/30% /O 2 : M3), and stored at 4 C. Quality evaluation was carried out using microbiological, chemical and sensory analyses. Microbiological results revealed that the M2 and VP delayed microbial growth compared with that of air-packaged samples. The effect was more pronounced for total viable count (TVC), Pseudomonas spp., lactic acid bacteria (LAB) and H 2 S-producing bacteria. TVC was reduced by 0Æ9 1Æ0, Pseudomonas spp. by 0Æ7 0Æ8, LAB by 1Æ0 2Æ2, H 2 S-producing bacteria by 0Æ7 1Æ2. Enterobacteriaceae were not significantly affected by MAP conditions. Of the chemical indices determined, the total volatile basic nitrogen and trimethylamine nitrogen values remained lower than the proposed acceptability limits of 35 mg N 100 g )1 and 12 mg N 100 g )1, respectively, after 15 days of storage. Both the VP and air-packaged mussel samples exceeded these limits. The thiobarbituric acid value of all MAP and VP mussels remained lower than the proposed acceptability limit of 1 mg malondialdehyde kg )1. The air-packaged samples exceeded this limit. All samples retained desirable sensory characteristics during the first 8 days of storage. Conclusions: Based on odour and taste evaluation, the M1 and M3 samples remained acceptable until ca day 11 12, the M2 samples remained acceptable until ca day 14 15 days while the VP and air-packaged mussel samples remained acceptable until ca days 10 11 and 8 9 of storage respectively. Based primarily on sensory, but also on biochemical and microbiological parameters determined, M2 gas mixture was the most effective for mussel preservation achieving a shelf-life of ca 14 15 days. Significance and Impact of the Study: MAP can be used to increase the shelf-life of refrigerated mussels. A shelf-life extension of refrigerated mussels by ca 5 6 days under MAP may be obtained. Keywords: biochemical, microbiological, modified atmosphere packaging, mussels, sensory analysis. Correspondence to: I.N. Savvaidis, Laboratory of Food Chemistry and Microbiology, Department of Chemistry, University of Ioannina, GR-45110 Ioannina, Greece (e-mail: isavvaid@cc.uoi.gr). ª 2004 The Society for Applied Microbiology

MICROBIOLOGICAL, BIOCHEMICAL AND SENSORY ASSESSMENT OF MUSSELS 753 INTRODUCTION Mussels have an exceptional nutritional value making them ideal for the human diet. Mussel flesh is rich in selenium, calcium, iron, magnesium, phosphorous and vitamins (A, B1, B2, B6, B12 and C) (Karakoltsidis et al. 1995; Vareltzis 1996). Mussel fat is also rich in polyunsaturated fatty acids (PUFA, 37 48% of total fatty acids) mainly x-3 PUFA (Orban et al. 2002). These fatty acids are biologically important and have been associated with a decreased risk of cardiovascular disease (Kromhout et al. 1985). The aquaculture production of mussels in Greece has rapidly increased over the past decade. Presently, Greece produces over 32 000 tons of mussels annually (Anonymous 2000). Mussels are usually marketed as raw, unshelled or shucked refrigerated products packaged in plastic pouches or frozen in vacuum sealed plastic pouches. They are also sold as processed products, i.e. smoked mussels. Currently, there is a growing tendency for consumption of fresh rather than frozen mussels owing to their high nutritional quality. The high activity (a w >0Æ95), high glycogen and free amino acids content and high ph (6Æ7 7Æ1) of mussels make them an ideal substrate for the growth of microorganisms (Jay 1996). Shelf-life of refrigerated mussels is 6 7 days. As a result of their very limited shelf-life, research on new preservation methods and shelf-life extension of such products is required. Modified atmosphere packaging (MAP) in combination with refrigeration has proved to be an effective preservation method for the extension of shelf-life and quality retention of a wide variety of fresh chilled food products (red meat, poultry, fruits, vegetables, bakery products, fresh pasta, fish and fish products) (Brody 1989; Brody and Marsh 1997; Davies 1997). The gas mixture used in a given MAP application must be chosen to meet the needs of the specific food product. Seafoods, unlike other muscle foods, are very susceptible to both microbiological and chemical deterioration. The shelflife of fish products in MAP can be extended depending on raw material, temperature, gas mixtures and packaging materials (Davies 1997). There are numerous studies in the literature on the effect of MAP on fish and fish products (Dalgaard et al. 1993; Dhananjaya and Stroud 1994; Bak et al. 1999; Hoz et al. 2000; Ruiz-Capillas and Moral 2001; Lopez-Caballero et al. 2002; Ozogul et al. 2004). Despite the work on preservation of fish using MAP, there is very limited information in the literature on the effect of MAP on the preservation of mussels. Thus, the objective of the present work was to study the effect of MAP including vacuum packaging on the shelf-life and quality of mussels stored at 4 C using microbiological, biochemical and sensory analyses. MATERIALS AND METHODS Preparation of mussel samples and storage conditions Aqua-cultured mussels (Mytilus galloprovincialis) were obtained from the harvest area of the culture farm (ALEX- ANDROS S.A.) in Macrigialos (Pieria, Greece) in April and June 2003. Mussels were transported to the laboratory within 4 h after harvesting in foamed polystyrene boxes containing ice. Mussels were subsequently inspected and dead animals were discarded. The remaining mussels were rapidly washed and the flesh meat was removed from the shell with a knife. All mussel flesh samples including the controls were packaged in low density polyethylene/polyamide/low density polyethylene barrier pouches (200 g per pouch) 75 lm in thickness having an oxygen permeability of 52Æ2 cm 3 m )2 day )1 atm )1 at 75% relative humidity (RH), 25 C and a vapour permeability of 2Æ4 gm )2 day )1 at 100% RH, 25 C. The following gas mixtures were used M1: 50%/50% ( ), M2: 80%/20% ( )and M3: 40%/30%/30% ( /O 2 ). Pouches were heatsealed using a HELMUT BOSS model N48 vacuum sealer (Bad Homburg, Germany) and kept under refrigeration (4 ± 0Æ5 C). Identical mussel samples were vacuum packaged. Control samples included mussels, air packaged with or without. Sampling was carried out at predetermined intervals namely 1, 3, 5, 8, 11 and 15 days. Microbiological analysis Mussel flesh (25 g) was transferred aseptically to a stomacher bag (Seward, Medical, UK) containing 225 ml of 0Æ1% peptone and homogenized for 60 s using a Lab Blender 400, Stomacher at room temperature (Seward Medical). For microbial enumeration, 0Æ1 ml samples of serial dilutions (1 : 10, diluent, 0Æ1% peptone ) of mussel homogenates were spread on the surface of dry media. Total viable counts (TVC) were determined using plate count agar (PCA; Merck, Darmstadt, Germany), after incubation for 2 days at 30 C. Pseudomonads were enumerated on cetrimide fusidin cephaloridine agar (CFC; Oxoid code CM 559, supplemented with SR 103, Oxoid, Basingstoke, UK) and incubated at 20 C for 2 days (Mead and Adams 1977). Lactic acid bacteria (LAB) were enumerated on de Man Rogosa Sharpe agar (MRS; ph 6Æ2, Oxoid code CM361, Oxoid) incubated at 25 C for 5 days. For Enterobacteriaceae and H 2 S-producing bacteria (including Shewanella putrefaciens) enumeration, a 1Æ0-ml sample was inoculated into 10 ml of molten (45 C) violet red bile glucose agar (VRBGA; Oxoid code CM 485, Oxoid) and Iron Agar (IA; Oxoid code CM 867, Oxoid)

754 A.E. GOULAS ET AL. respectively. After setting, a 10-ml overlay of molten medium was added. For the former (VRBGA; Oxoid), incubation was carried out at 30 C for 24 h. The large colonies with purple haloes were counted (Mossel et al. 1979). IA plates were incubated at 20 C and black colonies formed by the production of H 2 S were enumerated after 2 3 days. Three replicates of at least three appropriate dilutions depending on the sampling day were enumerated. Microbiological data were transformed into logarithms of the number of colony-forming units (CFU g )1 ). All plates were examined visually for typical colony types and morphology characteristics associated with each growth medium. Biochemical analysis Total volatile basic nitrogen (TVBN), trimethylamine nitrogen (TMAN) and 2-thiobarbituric acid (TBA) were determined according to the methods described previously (Goulas and Kontominas 2004). Sensory assessment The sensory quality of cooked mussels was evaluated at each sampling (1, 3, 5, 8, 11 and 15 days) by a seven member-trained panel. Mussel samples (140 g) were cooked individually in a microwave oven at full power for 2 min and immediately presented to the panelists (each panelist evaluating ca 20 g of mussel sample). Samples were presented to each panelist in plastic cups covered with a lid in random order. Panelists were asked to score odour, taste and appearance of cooked mussel and appearance of raw mussels using a 0 10 acceptability scale, where a score of 10 was defined as excellent. For each sensory attribute, a score of 6 (recorded by at least 50% of the judges) was considered to be the lower limit of acceptability, implying that shelf-life was terminated when this score was obtained. A freshly thawed mussel sample, stored at )30 C was also presented to the panelists, this serving as the master-control sample. Statistical analysis All analyses were run in triplicate (three different packaging samples) for each of two replicates (n ¼ 2 3). Results are reported as mean ± standard deviation (S.D.). Differences in mean values were determined using the Student s t-test method (significance was defined at P < 0Æ05). RESULTS Microbiological changes The present study focused on the monitoring of the following species of microorganisms: TVC, Pseudomonads, H 2 S-producing bacteria (including S. putrefaciens), Enterobacteriaceae and LAB. The initial TVC of mussels (Table 1) was ca 4Æ5 log CFU g )1 (day 1). TVC exceeded the value of 7 log CFU g )1, which is considered as the upper acceptability limit for fresh and marine species as defined by ICMSF (1986), on day 8 (M1 and M3), on day 11 (two control samples) and on day 15 (M2 and VP samples). After 15 days of storage, the M2 gas mixture and VP had a significantly lower (P <0Æ05) TVC count than the other two MAP and control samples. Initial population of Pseudomonads, Gram-negative contributor of natural flora of mussels, was ca 3Æ5 log CFU g )1 while a count of 7 log CFU g )1 was exceeded on day 8 and on day 11 (M1 and two control samples) (Table 2). After 15 days of storage, the M2 gas mixture and VP had a significantly lower (P <0Æ05) Pseudomonas spp. count (6Æ8 log CFU g )1 ) than all the rest of the samples. H 2 S-producing bacteria (including S. putrefaciens) were also a dominant bacterial species in mussel spoilage (Table 3). Initial H 2 S-producing bacteria count was ca 2Æ3 log CFU g )1, while a count of 7 log CFU g )1 was exceeded on day 11 (M1, M3 and the air/ control samples) and on day 15 (air control samples). The M2 gas /O 2 Air/ Table 1 Changes in total viable count* of refrigerated mussels packaged under various conditions 1 4Æ5 ±0Æ2 4Æ5 ±0Æ2 4Æ5 ±0Æ2 4Æ5 ±0Æ2 4Æ5 ±0Æ2 4Æ5 ±0Æ2 3 4Æ2 ±0Æ2 4Æ2 ±0Æ2 3Æ8 ±0Æ2 4Æ1 ±0Æ2 4Æ2 ±0Æ1 4Æ4 ±0Æ1 5 5Æ2 ±0Æ3 5Æ5 ±0Æ3 4Æ4 ±0Æ2 5Æ3 ±0Æ3 5Æ8 ±0Æ4 5Æ2 ±0Æ3 8 6Æ5 ±0Æ3 7Æ2 ±0Æ4 6Æ5 ±0Æ3 7Æ5 ±0Æ3 6Æ5 ±0Æ3 6Æ4 ±0Æ3 11 6Æ8 ±0Æ2 7Æ8 ±0Æ5 6Æ9 ±0Æ4 8Æ1 ±0Æ5 7Æ5 ±0Æ3 7Æ6 ±0Æ4 15 7Æ0 ±0Æ3 7Æ9 ±0Æ4 7Æ0 ±0Æ3 8Æ6 ±0Æ4 8Æ0 ±0Æ4 7Æ9 ±0Æ3 *Points represent mean values (log CFU g )1 ) of six determinations (n ¼ 2 3) ± S.D.

MICROBIOLOGICAL, BIOCHEMICAL AND SENSORY ASSESSMENT OF MUSSELS 755 Table 2 Changes in Pseudomonas spp.* of refrigerated mussels packaged under various conditions /O 2 Air/ 1 3Æ5 ±0Æ2 3Æ5 ±0Æ2 3Æ5 ±0Æ2 3Æ5 ±0Æ2 3Æ5 ±0Æ2 3Æ5 ±0Æ2 3 3Æ5 ±0Æ4 3Æ3 ±0Æ0 3Æ4 ±0Æ2 3Æ5 ±0Æ1 3Æ6 ±0Æ2 3Æ3 ±0Æ3 5 4Æ4 ±0Æ1 5Æ1 ±0Æ5 4Æ2 ±0Æ4 5Æ1 ±0Æ2 5Æ4 ±0Æ4 4Æ5 ±0Æ2 8 5Æ9 ±0Æ3 6Æ6 ±0Æ3 6Æ1 ±0Æ3 7Æ1 ±0Æ4 6Æ1 ±0Æ2 6Æ0 ±0Æ3 11 6Æ5 ±0Æ6 7Æ4 ±0Æ4 6Æ6 ±0Æ2 7Æ8 ±0Æ3 7Æ2 ±0Æ4 7Æ2 ±0Æ5 15 6Æ8 ±0Æ3 7Æ6 ±0Æ5 6Æ8 ±0Æ3 8Æ0 ±0Æ5 7Æ6 ±0Æ3 7Æ5 ±0Æ4 *Points represent mean values (log CFU g )1 ) of six determinations (n ¼ 2 3) ± S.D. mixture and VP sample did not reach this value throughout the 15-day storage period. LAB were also part of the natural flora of mussels (Table 4). Initial LAB count was ca 2Æ2 log CFU g )1, while a count of 7 log CFU g )1 was exceeded on day 11 (M3 and control 1) and on day 15 samples respectively. The M2 gas mixture-packaged samples did not reach this value throughout the 15-day storage period. The same observation holds for VP and without mussel samples. The M2 gas mixture had a significantly lower (P <0Æ05) LAB count than all packaged samples after 15 days of storage. Finally, Enterobacteriaceae produced counts lower (P < 0Æ05) than those for other microbial species in mussel samples. The initial Enterobacteriaceae count of 1Æ5 log CFU g )1 increased to 5Æ0 6Æ2 log CFU g )1 after 15 days of storage under all packaging conditions (results not shown). Biochemical analyses TVBN values of mussel samples are presented in Table 5. TVBN values of samples stored for 15 days in M1, M2 and M3 gas mixtures, respectively, remained significantly lower (P <0Æ05) than the acceptability limit of 35 mg N 100 g )1 of fish muscle set by the EEC (EEC 1995) and also proposed by Connell (1990) for fish-quality assessment. TVBN values of all other mussel samples exceeded the above limit. TMAN values are presented in Table 6. After 15 days of storage, TMAN values of all MAP-stored mussel samples were significantly lower (P < 0Æ05) than the proposed limit of acceptability for TMAN (12 mg N 100 g )1 ) for fish muscle, while the VP and control (with or without ) samples exceeded this limit on day 15 of storage. The MAP and VP samples produced significantly lower (P <0Æ05) TBA values in comparison with air-packaged mussel samples throughout the entire storage period. After 15 days of storage, the MAP and the VP samples produced statistically significant (P <0Æ05) lower TBA values than the rest of MAP samples. Finally, TBA values of all mussel VP and MAP mussel samples (0Æ63 1Æ60 mg malondialdehyde (MDA) kg )1 ) did not exceed the value of 1 2 mg MDA kg )1, regarded as the limit beyond which fish will normally develop an objectionable odour/taste (Connell 1990), although TBA values for the VP and MAP samples were lower than 1 mg MDA kg )1. Sensory analysis The results of the sensory evaluation of mussels (cooked and raw) showed that appearance scores for both cooked Table 3 Changes in H 2 S-producing bacteria (including Shewanella putrefaciens) count* of refrigerated mussels packaged under various conditions /O 2 Air/ 1 2Æ3 ±0Æ2 2Æ3 ±0Æ2 2Æ3 ±0Æ2 2Æ3 ±0Æ2 2Æ3 ±0Æ2 2Æ3 ±0Æ2 3 3Æ3 ±0Æ2 4Æ0 ±0Æ1 3Æ1 ±0Æ1 3Æ6 ±0Æ3 3Æ8 ±0Æ3 3Æ1 ±0Æ1 5 4Æ6 ±0Æ2 5Æ0 ±0Æ2 3Æ9 ±0Æ3 4Æ8 ±0Æ4 5Æ2 ±0Æ3 4Æ5 ±0Æ2 8 5Æ1 ±0Æ4 6Æ0 ±0Æ2 6Æ2 ±0Æ4 6Æ7 ±0Æ5 6Æ0 ±0Æ4 5Æ9 ±0Æ3 11 6Æ1 ±0Æ2 7Æ1 ±0Æ3 6Æ4 ±0Æ4 7Æ8 ±0Æ6 7Æ1 ±0Æ3 6Æ9 ±0Æ4 15 6Æ4 ±0Æ4 7Æ4 ±0Æ3 6Æ8 ±0Æ3 8Æ0 ±0Æ4 7Æ5 ±0Æ5 7Æ6 ±0Æ4 *Points represent mean values (log CFU g )1 ) of six determinations (n ¼ 2 3) ± S.D.

756 A.E. GOULAS ET AL. /O 2 Air/ Table 4 Changes in lactic acid bacteria count* of refrigerated mussels packaged under various conditions 1 2Æ2 ±0Æ2 2Æ2 ±0Æ2 2Æ2 ±0Æ2 2Æ2 ±0Æ2 2Æ2 ±0Æ2 2Æ2 ±0Æ2 3 2Æ5 ±0Æ1 3Æ1 ±0Æ1 2Æ9 ±0Æ2 2Æ6 ±0Æ1 3Æ1 ±0Æ1 2Æ5 ±0Æ5 5 3Æ4 ±0Æ2 4Æ9 ±0Æ2 3Æ8 ±0Æ1 4Æ6 ±0Æ1 4Æ5 ±0Æ2 2Æ7 ±0Æ4 8 4Æ5 ±0Æ2 6Æ0 ±0Æ2 4Æ5 ±0Æ2 5Æ7 ±0Æ5 6Æ0 ±0Æ4 4Æ7 ±0Æ3 11 5Æ3 ±0Æ1 6Æ6 ±0Æ3 4Æ8 ±0Æ3 7Æ1 ±0Æ4 7Æ2 ±0Æ3 5Æ6 ±0Æ4 15 6Æ3 ±0Æ4 6Æ9 ±0Æ4 5Æ2 ±0Æ2 7Æ3 ±0Æ3 7Æ4 ±0Æ2 6Æ2 ±0Æ3 *Points represent mean values (log CFU g )1 ) of six determinations (n ¼ 2 3) ± S.D. /O 2 Air/ Table 5 Total volatile basic nitrogen* (TVBN) (mg N 100 g )1 ) of mussels stored at 4±0Æ5 C 1 11Æ48 ± 0Æ34 11Æ48 ± 0Æ34 11Æ48 ± 0Æ34 11Æ48 ± 0Æ34 11Æ48 ± 0Æ34 11Æ48 ± 0Æ34 3 13Æ58 ± 0Æ47 12Æ18 ± 0Æ21 11Æ76 ± 0Æ24 11Æ62 ± 0Æ40 11Æ48 ± 0Æ27 13Æ58 ± 0Æ35 5 16Æ10 ± 0Æ34 12Æ88 ± 0Æ29 12Æ88 ± 0Æ51 12Æ60 ± 0Æ35 12Æ92 ± 0Æ32 17Æ78 ± 0Æ61 8 18Æ90 ± 0Æ22 19Æ08 ± 0Æ47 14Æ73 ± 0Æ46 19Æ59 ± 0Æ43 23Æ10 ± 0Æ25 21Æ60 ± 0Æ53 11 29Æ40 ± 0Æ41 22Æ42 ± 0Æ55 17Æ54 ± 0Æ35 25Æ23 ± 0Æ26 29Æ41 ± 0Æ44 35Æ66 ± 0Æ71 15 39Æ25 ± 0Æ25 30Æ10 ± 0Æ36 25Æ22 ± 0Æ43 28Æ79 ± 0Æ68 36Æ72 ± 0Æ62 45Æ80 ± 0Æ59 *Values represent the mean of six determinations (n ¼ 2 3) ± S.D. /O 2 Air/ Table 6 Trimethylamine nitrogen* (TMAN) (mg N 100 g )1 ) of mussels stored at 4±0Æ5 C 1 1Æ82 ± 0Æ09 1Æ82 ± 0Æ09 1Æ82 ± 0Æ09 1Æ82 ± 0Æ09 1Æ82 ± 0Æ09 1Æ82 ± 0Æ09 3 3Æ02 ± 0Æ17 2Æ11 ± 0Æ06 2Æ05 ± 0Æ05 2Æ69 ± 0Æ18 3Æ15 ± 0Æ12 3Æ58 ± 0Æ23 5 4Æ84 ± 0Æ20 3Æ36 ± 0Æ12 2Æ82 ± 0Æ14 3Æ10 ± 0Æ15 4Æ45 ± 0Æ25 4Æ80 ± 0Æ19 8 6Æ79 ± 0Æ17 4Æ81 ± 0Æ28 4Æ20 ± 0Æ23 5Æ02 ± 0Æ40 5Æ97 ± 0Æ20 6Æ46 ± 0Æ30 11 9Æ24 ± 0Æ26 6Æ80 ± 0Æ34 5Æ93 ± 0Æ12 6Æ48 ± 0Æ26 8Æ26 ± 0Æ37 8Æ59 ± 0Æ29 15 14Æ88 ± 0Æ41 8Æ99 ± 0Æ31 7Æ87 ± 0Æ25 8Æ48 ± 0Æ33 12Æ44 ± 0Æ29 13Æ69 ± 0Æ47 *Values represent the mean of six determinations (n ¼ 2 3) ± S.D. and raw samples decreased at a slower rate than odour and taste scores (results not shown). In general, the appearance of the raw mussel samples received a lower (P <0Æ05) score than appearance of cooked mussel samples. All mussel samples received a score of ÔexcellentÕ (9 10) or Ôvery goodõ (7 8) during the first 5 days with regard to odour and taste and during the first 8 days with regard to appearance. After this period, significant differences (P <0Æ05) were observed in sensory scores of VP, MAP and control samples. The limit of acceptability (score 6) for odour and taste was exceeded on day 11 (VP, two control samples) and on day 15 (M1, M2 and M3) samples respectively. DISCUSSION In the present study initial TVC for mussels indicate acceptable quality, given that the upper acceptable limit for TVC of mussels is 5 10 5 CFU g )1 (ICMSF 1986). Similarly, Pastoriza et al. (2004) reported a TVC value of 3Æ88 log CFU g )1 for mussels on day 1 after harvesting. Of

MICROBIOLOGICAL, BIOCHEMICAL AND SENSORY ASSESSMENT OF MUSSELS 757 the three gas atmospheres, the ( ) gas mixture was the most effective for the inhibition of TVC. This fact may be attributed to the inhibitory effect of the higher concentration of CO 2 (80%) on microbial growth. CO 2, because of its bacteriostatic effect, inhibits the growth of aerobic Gram-negative bacteria such as Pseudomonas spp. and Shewanella spp. as a result of an extension of lag phase of growth, and a decrease in the growth rate during the logarithmic phase (Farber 1991; Debevere and Boskou 1996; Gram and Huss 1996; Sivertsvik et al. 2002). Similar effects of MAP have been reported for various marine species. Ozogul et al. (2004) reported that TVC grew most quickly in sardines (Sardina pilchardus) stored in air, followed by those in VP while the lowest counts were obtained with MAP (60% CO 2 and 40% N 2 ). According to Lopez- Caballero et al. (2002), who studied the effect of MAP on the microbial flora of pink shrimp (Parapenaeus longirostris), TVC in MAP- [CO 2 /O 2 /N 2 (%): and 45/5/50] stored shrimp was lower by two log cycles than those for airstored shrimp at the end of storage period. Microbial counts of filleted salmon (Salmo salar) were clearly higher in airpacked and slightly higher in VP samples than in gas-packed samples (Randell et al. 1999). Based on a microbiological acceptability limit of 7 log CFU g )1 for fresh and marine species (ICMSF 1986) and TVC data of the present study, MAP and VP resulted in an extension of shelflife of mussels by ca 5 days. In other studies, Hoz et al. (2000) reported that a CO 2 /air (40/60, v/v) atmosphere was the most effective in extending the shelf-life of refrigerated salmon, whereas the shelf-life of sea bass slices packaged in 80 100% CO 2 atmosphere was extended to more than 20 days at 4 C compared with 9 days for those packaged in air (Masniyom et al. 2002). Finally, oregano oil (0Æ05%) reduced growth of Photobacterium phosphoreum in naturally contaminated MAP cod fillets and extended shelf-life from 11 12 days to 21 26 days at 2 C (Mejlholm and Dalgaard 2002). It is well documented that Pseudomonas and Shewanella spp. grow during storage on chilled fish and seafood products (Gram and Dalgaard 2002), and that both species are major contributors to mussel spoilage (Jay 1996). Counts of H 2 S-producing bacteria (of which S. putrefaciens is the predominant species) and Pseudomonads have been used as indicators of spoilage of iced fish, fish products and oysters (Dalgaard et al. 1993; Gram and Huss 1996; Hoz et al. 2000; Lopez-Caballero et al. 2000; Kyrana and Lougovois 2002; Mendes et al. 2002). Especially, the S. putrefaciens count is directly related to remaining shelf-life of fish causing reduction of trimethylamine oxide (TMAO) to TMA in fish (Debevere and Boskou 1996; Gram and Huss 1996). Surprisingly in the present study, Pseudomonas spp., being psychrotrophic and strictly aerobic bacteria, grew in refrigerated mussels irrespective of the packaging atmosphere conditions; their populations were the highest in the M3 gas mixture as expected because of its higher O 2 content (30%). It must be stated, however, that to the best of our knowledge no other work has been found in the literature dealing with the monitoring of the growth of Pseudomonas spp. in mussels stored under MAP and VP conditions, in contrast to studies dealing with preservation of fresh fish and seafood products stored in ice. With regard to H 2 S-producing bacteria (including Shewanella spp.), growth was partly inhibited in mussels kept under MAP conditions. Likewise, Lopez-Caballero et al. (2002) found that MAP [CO 2 /O 2 /N 2 (%): and 45/5/ 50] inhibited the growth of H 2 S-producing bacteria in shrimp. LAB were found to be members of the final microbial flora of MAP, VP and air-packaged samples (control samples), and may also play an active role in the spoilage of the mussels. Similarly under MAP conditions, LAB growth was also observed in hake steaks (Ordonez et al. 2000) and in refrigerated sea bass (Masniyom et al. 2002). LAB inhibit growth of other bacteria because of the formation of lactic acid and bacteriocins, and this may contribute to their selective growth during spoilage of seafood products. Enterobacteriaceae count of fresh mussels in the present study is lower than that reported by other investigators for other fish species: 1Æ9 log CFU g )1 for sea bream (Koutsoumanis et al. 1999), 1Æ9 log CFU g )1 for shrimp (Lopez- Caballero et al. 2002) and 2Æ0 log CFU g )1 for sea bass (Taliadourou et al. 2003). Enterobacteriaceae followed a similar trend to LAB and their counts were lower compared with those of other microbial species, in agreement with results obtained by Ordonez et al. (2000) and Lopez- Caballero et al. (2002) for hake steaks and for shrimp stored under CO 2 /O 2 -modified atmospheres respectively. Enterobacteriaceae being facultative anaerobes psychrotrophic in nature may grow in refrigerated foods in significant numbers, irrespective of the specific packaging conditions (VP, MAP or air) as shown in the present study. The presence of Enterobacteriaceae in the microflora of marine species and their spoilage potential must be taken into consideration especially in the case of polluted aquatic environment, given that mussel is a -filtering organism. Initial TVBN value of mussel samples, 1 day after harvesting (11Æ48 mg N 100 g )1 ), is indicative of freshness of raw material and is in good agreement with the initial value of 11Æ2 mg N 100 g )1, reported by Lopez-Caballero et al. (2000) for fresh oysters (Osttraca edulis). However, Pastoriza et al. (2004) reported a TVBN value of 8Æ47 mg N 100 g )1 for mussels, 1 day after harvesting. Observing the TVBN values of all mussels samples, the corresponding odour and taste scores of cooked samples, and given the rejection sensory score of 6, it is proposed that a

758 A.E. GOULAS ET AL. more realistic TVBN limit for mussels is ca 22 25 mg N 100 g )1 as compared with the value of 35 mg N 100 g )1 proposed for fish (Connell 1990). This proposed value is somewhat lower than that reported by Lopez-Caballero et al. (2000) (25 30 mg N 100 g )1 ) for the spoilage for oysters. A possible reason for a relatively low TVBN content at the of spoilage may be that mussels undergo a general acidification because of their high glycogen content which is converted to lactic acid (Lopez- Caballero et al. 2000). Given that TVBN value is the result of titration with HCl, the lactic acid produced neutralized part of the base (TVBN) and thus reduced the calculated TVBN values. According to Orban et al. (2002), the glycogen content of mussels (Mytilus galloprovincialis) is 12Æ7 24Æ8% on a dry basis. The gas mixture () was the most effective for mussel preservation producing the lowest (P <0Æ05) TVBN value (25Æ22 mg N 100 g )1 ), after 15 days of storage. This gas mixture contributes to the extension of the shelf-life of mussels to ca 15 days as compared with both control mussel samples which exceeded the value of 25 mg N 100 g )1 on day 11 of storage. Present results are in agreement with those of Kim et al. (2002) who reported a TVBN value of 42 mg N 100 g )1 for air packed oyster samples after 15 days of storage at 3 C. TMA is produced by the decomposition of TMAO caused by bacterial action and possibly through the action of intrinsic enzymes (Connell 1990; Reddy et al. 1997). The highest concentration of TMAN was observed for the mussel samples packaged under vacuum, followed by mussels stored in air and lastly by samples under MAP. This is in agreement with results reported by other authors, according to which anaerobic conditions enhance TMA production as a result of fish spoilage bacterial action on TMAO (Debevere and Boskou 1996; Gram and Huss 1996; Valle et al. 1999). Of course TMAN content varies with species, season and type of storage (Connell 1990; Ababouch et al. 1996; Reddy et al. 1997). Lower TMAN values in MAP samples can be attributed to bacterial growth inhibition by CO 2 as well as to the partial change in microbial flora when marine products are stored in enriched CO 2 atmospheres (Boskou and Debevere 1997; Hoz et al. 2000; Ruiz-Capillas and Moral 2001). The TBA index is a measure of MDA, one of the degradation products of lipid hydroperoxides, formed from PUFA (Raharjo and Sofos 1993). As can be seen in Table 7, there is a trend towards an increase in TBA values up to a certain point during the storage period, followed by either a decrease in these values or a lower increase rate. Decrease in TBA values may be caused by interaction between MDA and proteins, aminoacids, glycogen, etc. resulting in lower amount of free MDA. This observation is in agreement with results reported by other authors (Curzio and Quaranta 1982; Fernandez et al. 1997; Ruiz-Capillas and Moral 2001; Chouliara et al. 2004; Goulas and Kontominas 2004). The highest sensory scores were observed for mussel samples packaged under MAP with higher CO 2 concentration (80% CO 2 /20%N 2 ). This is in agreement with results reported by Pastoriza et al. (2004) for mussels packaged in 75% CO 2 /25% N 2. Based on odour and taste scores, a shelf-life of ca 14 15 days was achieved for mussel samples packaged under M2 gas mixture. This gas composition extended the shelflife of mussels for ca 5 6 days, as compared with the control samples, retained higher sensory scores than all other packaged samples (P <0Æ05), while the other two MAP used extended the shelf-life of mussels by ca 3 days. Odour and taste data of packaged mussels correlated rather well with microbiological data (TVC) with the exception of M1- and M3-packaged mussel samples. Considering the proposed microbiological limit of acceptability (7 log CFU g )1 ), a shelf-life of ca 15 days for the VP and M2 mussel samples, 10 11 days for two control samples and 8 9 days for M1 and M3 samples was achieved. The discrepancy observed between sensory and microbiological data was ca 4 days for VP and 6 7 days for M1 and M3 /O 2 Air/ Table 7 Thiobarbituric acid* (TBA) (mg malondialdehyde kg )1 ) of mussels stored at 4 ± 0Æ5 C 1 0Æ31 ± 0Æ06 0Æ31 ± 0Æ06 0Æ31 ± 0Æ06 0Æ31 ± 0Æ06 0Æ31 ± 0Æ06 0Æ31 ± 0Æ06 3 0Æ55 ± 0Æ10 0Æ39 ± 0Æ15 0Æ38 ± 0Æ10 0Æ48 ± 0Æ08 0Æ63 ± 0Æ07 0Æ70 ± 0Æ11 5 0Æ47 ± 0Æ09 0Æ71 ± 0Æ05 0Æ80 ± 0Æ07 0Æ82 ± 0Æ14 1Æ07 ± 0Æ06 1Æ27 ± 0Æ15 8 0Æ67 ± 0Æ06 0Æ62 ± 0Æ07 0Æ74 ± 0Æ14 0Æ88 ± 0Æ18 1Æ04 ± 0Æ05 1Æ10 ± 0Æ07 11 0Æ72 ± 0Æ13 0Æ54 ± 0Æ12 0Æ49 ± 0Æ08 0Æ85 ± 0Æ14 1Æ33 ± 0Æ24 1Æ20 ± 0Æ16 15 0Æ65 ± 0Æ08 0Æ79 ± 0Æ05 0Æ63 ± 0Æ10 0Æ93 ± 0Æ10 1Æ60 ± 0Æ15 1Æ46 ± 0Æ15 *Values represent the mean of six determinations (n ¼ 2 3) ± S.D.

MICROBIOLOGICAL, BIOCHEMICAL AND SENSORY ASSESSMENT OF MUSSELS 759 samples. Similar discrepancies between sensory and microbiological data have been reported by many authors (Hansen et al. 1995; Mendes et al. 2002; Ozogul et al. 2004) for coldsmoked salmon, pink shrimp and sardines, respectively and is owed to differences in populations between total and specific spoilage microorganisms. It should be stressed that particular attention must be given to process treatments involving MAP in the absence of oxygen in view of the possibility of growth and toxin production of Clostridium botulinum type E under prolonged storage conditions. at temperatures below 3 C can preclude this possibility, but one cannot guarantee against temperature abuse once a product enters the distribution channel. In conclusion, based on sensorial data, the shelf-life of mussel samples was 11 12 days (M1 and M3), 14 15 days, 10 11 days and 8 9 days (control samples). 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