Abstract The microflora of farmed gilthead seabreamstored in ice and the effect of washing of the fish withtap water were examined in different culture media andincubation temperatures. Washing reduced the number ofmicroorganisms in all culture conditions studied. For un-washed and washed lots the highest number of bacteriawere obtained on spread plates in Iron Agar + 0.5%NaCl incubated at 15 °C, H2S-producing microorgan-isms constituting a high percentage of the total flora. Thecorrelation, up to the sensory rejection point, betweenbacterial counts and sensory score for the raw fish variedwith washing and with the culture conditions, and washigher for the H2S-producing microorganisms.

Keywords Gilthead seabream · Washing · Microflora ·Culture conditions

Introduction

The flesh of live and healthy fish is sterile. The principalcontamination of fish after death is associated with theflora present in skin, gills and guts and is related to theconditions of the water where the fish live. Bacterialgrowth during storage increases with handling. A100–1000-fold increase of the number of bacteria hasbeen found on the fish (mainly in the mucus) immediate-ly after direct contact with decks, equipment and boxes[1, 2]. Marine fish mucus is a glycoprotein solution con-taining sugars, amino sugars and free amino acids. Theantibiotic properties, or even the antibodies, of the mu-cus can afford some protection to the live fish and also tothe fish flesh for a few days after death, but its removalshould prevent spoilage in view of the large number ofbacteria that it contains [1]. Microorganisms are thechief cause of spoilage during ice storage [3, 4]. Effi-

cient washing of the fish before and/or after processingor during chilled storage will have a decisive effect onshelf life because washing takes away a proportion of themicroorganisms on the outer surface of the fish thus re-ducing the number of bacteria capable of penetrating themuscle by diffusion [1, 5]. Washing of the fish may oc-cur during rapid chilling (refrigerated sea water [RSW],chilled sea water [CSW], liquid ice, etc.), by melt waterduring storage in ice, or by washing with tap water,which is occasionally done at points of sale where it issometimes standard commercial practice. Washing doesnot eliminate the entire flora present because the com-mensal population specific to fish is better adapted tosurvive in the mucus, whereas the contaminant florafrom ice, equipment, etc. is more readily eliminated [6,7]. For years there was strong opposition to washing offish taking into account the protective effect of the mu-cus. However, it was also demonstrated that eliminationof the mucus prior to storage of fish prolonged shelf lifebecause a considerable microbial load can develop in themucus [8].

Several criteria are currently used in determining mi-croorganisms in fish (temperature, salinity of the medi-um, etc). Traditionally, the total bacterial load in fish hasbeen expressed as counts on plate count agar incubatedat 30 °C [9], although for fish or fish products some au-thors apply other conditions such as lower incubationtemperatures, 20 °C [2, 10, 11] or 15 °C [3, 12], otherculture media [11], different concentrations of NaCl andtype of plating [3, 12, 13].

Some authors consider that there is no correlation be-tween total number of bacteria and spoilage of fish, sincethe specific spoilage microorganisms (SSO) make up on-ly a fraction of the total microflora [14]. Total viablecounts at 20 °C have been found to give an indication ofthe hygiene maintained during the handling of fish, butthis appears to be a doubtful figure in terms of express-ing quality and predicting keeping time [2].

Although there are several references in the literaturedealing with quality and shelf life of gilthead seabream[15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27], no da-

M.E. López-Caballero · A. Huidobro · A. Pastor · M. Tejada (✉)Instituto del Frío (CSIC). Ciudad Universitaria s/n. 28040 Madrid,Spaine-mail: mtejada@if.csic.esTel.: 34 91 544 2300, Fax: 34 91 549 3627

Eur Food Res Technol (2002) 215:396–400DOI 10.1007/s00217-002-0574-2

O R I G I N A L PA P E R

María Elvira López-CaballeroAlmudena Huidobro · Ana Pastor · Margarita Tejada

Microflora of gilthead seabream (Sparus aurata) stored in ice. Effect of washing

Received: 12 February 2002 / Revised: 31 May 2002 / Published online: 16 August 2002© Springer-Verlag 2002

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ta were found on the different culture conditions as theyrelate to bacterial load and sensory quality. Previousstudies on the effect of washing on the evolution of sen-sory quality in gilthead seabream measured by the quali-ty index method (QIM) has shown that washing modifiesthe evolution of the parameters awarded to this species.Washing reduces the demerit points assigned to this spe-cies during chilled storage and, as a result, the maximumdemerit point assigned to the QIM was not reached evenwhen the cooked fish was rejected by the taste panel.Washing also diminished the total viable count on plateagar at 30 °C [16].

As total counts may differ according to the culturemedium and/or incubation temperature chosen, we there-fore considered it useful to examine the microbial floraof gilthead seabream (Sparus aurata), unwashed and dai-ly washed with tap water using different culture condi-tions in order to ascertain which one relates better withthe sensory score.

Materials and methods

Sample preparation

A total of 80 kg of immature gilthead seabream (Sparus aurata)fasted for 48 h was obtained from a Spanish fish farm (Cupimar,San Fernando, Cádiz, Spain) in June 1999 and killed by immer-sion in an ice slurry. Immediately after death, the fish were packedin expanded polystyrene boxes with perforated bottoms, coveredby a perforated plastic film with ice flakes on top, sealed, andfreighted to the laboratory in refrigerated trucks. At the laboratory,the fish were divided into two lots and kept in boxes with ice incold stores at 2±1 °C for up to 22 days. In the control lot (un-washed fish), ice was added to the boxes as required. In thewashed lot, the fish was washed daily with tap water then storedagain with ice in the same conditions as the unwashed lot. Themean and standard deviation of the weight and length of the fishstudied were 261.73±27.55 g and 21.13±1.03 cm, respectively.

Microbiological analysis

From each lot, several portions of muscle with skin from differentparts of at least five individuals were removed aseptically in a ver-tical laminar-flow cabinet (Telstar mod. AV 30/70, Madrid,Spain), and mixed. Into a sterile plastic bag (Sterilin, Stone, Staf-fordshire, U.K.) was placed 10 g of the mixture and 90 ml of buf-fered peptone water (Oxoid, Basingstoke, UK) at 6±1 °C. After1 min in a Stomacher blender (mod. Colworth 400, Seward, Lon-don, UK), dilutions were made in the same diluent. The total num-ber of bacteria were determined as follows: pour plates of platecount agar (PCA, Oxoid) incubated at 30 °C for 72 h; pour platesof plate count agar (PCA, Oxoid) incubated at 20 °C for 72 h;double-layered plates of Iron Agar (IA, Adsa-Micro, Barcelona,Spain) incubated at 20 °C for 72 h; spread-plates of Iron Agar(Adsa-Micro) + 0.5% NaCl incubated at 15 °C for 5 days. TheH2S-producing microorganisms were determined as black colonieson double-layered plates of Iron Agar (Adsa-Micro) incubated at20 °C for 72 h and on spread-plates of Iron Agar (Adsa-Micro) +0.5% NaCl incubated at 15 °C for 5 days. Microbiological countswere expressed as log colony-forming units per gram of sample(log cfu/g). Microbiological analyses were performed in duplicate.

Sensory analyses

Sensory evaluation of the washed and unwashed fish was carriedout by applying the quality index method (QIM), developed forthis species by Huidobro et al. [21] and by a quality test in thecooked samples. The sensory results for these lots have been al-ready published [16].

Statistical analyses

Two-way analysis of variance with an F test and least-squares dif-ferences in means between pairs were used to examine, at differ-ent culture conditions, the lots at storage time and to determine theeffect of storage time on each lot. Data analysis was performed us-ing the Statistica 5.1 program (StatSoft Inc., Tulsa, OK, USA).

Results and discussion

The initial total bacteria counts were 3–4 log cfu/g de-pending on culture conditions and medium (Fig. 1). Aparallel microbial growth was found in both unwashedand washed lots during storage, although washing re-duced the total counts in 1 or 2 log units. Reductions ofup to 40% in total flora following washing of culturedfish have been reported [28]. Some authors reported thatlight washing of the control lot occasionally caused ir-regularity in counts, but the significant reduction of thebacterial load in the washed lot did prolong the shelf lifeof the fish [28, 29]. In our work, the unwashed lot at-tained or exceeded 7 log cfu/g between 11 and 14 daysof storage in most of the assayed culture conditions. Inthe washed lot, these counts were reached later (ataround day 17 of storage) except in IA at 15 °C that wasreached around day 14 (Fig. 1, Table 1). At the end ofthe storage period, the counts in unwashed fish reached8–9 log cfu/g. In all cases, counts were higher than in thewashed lot. The values recorded in unwashed fish werewithin the range reported for fillets of gilthead seabreamstored for 10 days at 0 °C, although the culture condi-tions were different (Long and Hammer Agar incubated

Fig. 1 Total bacteria count (log cfu/g) for raw chilled giltheadseabream unwashed (—) and daily washed with tap water (- -).Counts on pour plates of Iron Agar (IA) + 0.5% NaCl incubated at15 °C/5 days (diamonds) and Iron Agar (IA) incubated at 20 °C/3days (triangles), pour plates of plate count agar (PCA) incubatedat 20 °C/3 days (squares) and pour plates of plate count agar(PCA) incubated at 30 °C/3 days (circles)

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at 10 °C for 7 days) [15]. Drosinos and Nychas [18] re-ported total counts of 1010 cfu/g after 10 days in storagefor gilthead seabream fillets stored at 0±1 °C (PCA incu-bated at 20 °C for 4 days). In other cultured speciesSoultos et al. [30] found that the counts in iced storedsea bass did not exceed 7 log cfu/g even after 20 days instorage (Iron Agar at 25 °C for 3 days).

Initial counts of H2S-producing microorganisms werebelow the detection threshold (<1 and <2 log cfu/g in IAincubated at 20 °C and 15 °C respectively. Fig. 2). Kout-soumanis et al. [15] reported initial counts of about 3 logcfu/g of H2S-producers in iced gilthead seabream fillets,rising rapidly to 7 log cfu/g before the sensory rejectionlimit of the cooked fish (15 days) was reached. In thepresent study, the number of H2S-producers in giltheadseabream had risen to 7–8 log cfu/g by the time the sen-sory rejection limit was reached. Up to this point thecounts were also significantly higher in the unwashed lotfor the same culture conditions (Fig. 2, Table 2). The re-sults show that H2S-producers constituted a very highpercentage of the total flora (Figs. 1 and 2) which is con-sistent with the results reported by Soultos et al. [30].The spoilage association developed in aerobically storedfish typically consisted of gram-negative psychrotrophicnon-fermentative rods, mainly Pseudomonas and She-

wanella putrefaciens [14]. This observation is consistentwith results reported for chilled Mediterranean giltheadseabream [15, 18, 19, 23]. Although no specific mediumwas used to determine Pseudomonas spp in this study,the percentage of the total flora that did not produce H2Smay be tentatively considered as pseudomonads.

As regards the culture conditions (media and tempera-ture), the results showed differences in counts dependingon the chosen conditions, particularly in the early stagesof storage. The initial counts for gilthead seabream ac-cording to culture medium were one log unit less in pourplates of Iron Agar (IA) than in pour plates of plate countagar (PCA), both incubated at 20 °C (Fig. 1). As to incu-bation temperature, there were initially no differences inthe numbers of microorganisms in Iron Agar on pour orspread plates incubated at 20 and 15 °C, respectively noron PCA incubated at 20 and 30 °C (Fig. 1, Table 1). Theseinitial counts are comparable to those reported in giltheadseabream (pour plates of plate count agar at 32–35±1 °Cfor 2 days) by Abdalla et al. [17] and in farmed Europeansea bass by Soultos et al. [30]. Slightly higher values(4.5 log cfu/g) were found in gilthead seabream [15].

The microbiological counts in different culture mediaat the time of sensory rejection of the cooked fish showsthat the highest counts occurred in spread plates of IronAgar incubated at 15 °C (Fig. 1, Table 1). This was espe-cially evident at around 15 days of storage, which wasthe set limit for shelf life of these lots by sensory analy-sis of the raw fish [16]. Bachhil [28] found that in fishstored at 0–1 °C, the percent increase was higher in thepsychrophile (plate count agar incubated at 5 °C for 5–7days), than in the mesophile (plate count agar incubated37 °C for 24 h) group which is consistent with the resultsof the present study. Spread plates of Iron Agar + 0.5NaCl at 15 °C were chosen because some microorgan-isms cannot grow at the higher temperatures and lowerNaCl concentrations normally used [12]. The results in-dicate that during storage in ice the predominant generashows psychrotrophic characteristics which could play arole in fish spoilage and which register less on the platethe higher the incubation temperature.

The fact that the total load varies according to the cul-ture conditions is especially significant in ranges close to

Fig. 2 H2S-producing microorganisms (log cfu/g) for raw chilledgilthead seabream unwashed (—) and daily washed with tap water(- -). Counts on spread plates of Iron Agar (IA) + 0.5% NaCl incu-bated at 15 °C/5 days (diamonds) and on pour plates of Iron Agar(IA) incubated at 20 °C/3 days (triangles)

Table 1 Statistical analyses of total counts cultured in different conditions

Culture conditions Lots Days of storage

1 4 9 11 14 16 18 21

Total viable bacteria (Iron Agar at 15 °C) Unwashed a1 c2 a3 c4 c5 c6 c6 d6Washed a1 a1 b2 ab3 a4 d5 be5,6 be6

Total viable bacteria (Iron Agar at 20 °C) Unwashed a1 a1 a2 a3 a4 a4 a5 ad5Washed a1 b2 b3 b4 be5 b5 b6 b6

Total viable bacteria (PCA at 20 °C) Unwashed b1 d2 a3 d4 b5 a6 ae7 a8Washed b1 a2 d3 d4 d5 be6 d7 c7

Total viable bacteria (PCA at 30 °C) Unwashed b1 d2 c3 e4 e5 a6 be7 ad8Washed b1 a2 e1 f3 f4 e5 d6 ce7

Different letters in the same column indicate significant differences among culture conditions and different numbers in the same row in-dicate significant differences during the storage time (p≤0.05)

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the generally accepted microbiological limits, despite thefact that in advanced stages of storage the counts weresimilar in all culture media (Fig. 1). Considering the rec-ommended microbiological limit for the total count infresh fish (107 cfu/g) [31], the counts in the washed lotexceeded that limit at 16–18 days of storage in PCA at30 °C, that is several days later than in unwashed lot(Fig. 1, Table 1).

Huss et al. [2] found that the correlation between sen-sory score and total number of bacteria in the fish wasrather poor during storage irrespective of the bacterialload, and only beyond 10–11 days did the bacterial num-ber significantly influence the sensory score. Gillespieand Ostovar [8] reported that the microbial load was re-duced by one log unit by washing of the fish, althoughthey noted that the unwashed lot scored better in inspec-tion during the first three days of storage in ice at 3 °C.Thereafter, scores for washed fish changed little, where-as those for unwashed fish rapidly declined. In contrast,Batchhil [28] reported that washed fish presented betterappearance and acceptability despite the fact that after10 days of storage in ice they showed no signs of spoil-age. In the present case, sensory differences in the rawfish were detected after day 11 of storage in ice, assign-ing to both lots at this time 9 demerit points, applyingthe quality index method (QIM) [16]. Up to day 11 ofstorage, washing significantly reduced the microbialload, probably because part of the bacteria in the surfacewas eliminated. Differences in the microbial loads be-tween the two lots diminished afterwards.

Sensory rejection of the control lot applying QIM oc-curred at 15 days of storage [16]. At this point total bacte-ria count incubated at 15 °C registered 8–9 log cfu/g andH2S-producers about 8 log cfu/g (Figs. 1 and 2, respective-ly). Our results are in agreement with Koutsoumanis et al.[15] who found sensory differences in gilthead seabreamstored at 0 °C when the pseudomonads and H2S-producingmicroorganisms reached 106-107 cfu/g. Counts in the otherculture media for the same lot were lower. Koutsoumanisand Nychas [23] concluded that a spoilage model for aero-bically stored seabream can be based on pseudomonadgrowth (cetrimide fusidin cephaloridine agar incubated20 °C for 2 days), proposing the level of 107 cfu/g in thisgenus as a good determinant for the end of shelf life.

Sensory evaluation of the raw fish applying QIMshowed that the washed lot got lower demerit points than

the unwashed ones after 11 days of storage although thesensory rejection for the cooked fish was around 15 daysfor both lots [16]. This may be due to the fact that up to11 days of storage, lower demerit points were detected inparameters such as shape and clarity of eyes that werecompensated in the overall QIM score with the higherdemerit points for elasticity as result of washing [16].The differences in odour of fish (3 demerit points) andodour of gills (3 demerit points), that are the most impor-tant parameters in the QIM designed for gilthead sea-bream [21], produced a significant difference in the totalscore (maximum 15 demerit points) after 11 days of stor-age. Nevertheless in the sensory evaluation of thecooked filets, the rejection point was reached at 15 daysin both washed and unwashed fish, mainly because off-flavours were detected by the assessors. Taking into ac-count that at this time the total viable counts and H2Sproducers were ≥107 cfu/g in both lots, the lack of corre-spondence at the rejection point of both sensory methodswas probably due to the fact that cooking enhanced theperception of off-flavours.

A high correlation of bacterial counts versus sensoryscore measured as QIM up to the rejection point was ob-tained in IA at 15 °C for the unwashed and washed lot(unwashed: y=0.4149x+3.4154 R2=0.9795; washed:y=0.5136x+2.1889 R2=0.9109). Nevertheless, the corre-lation slopes of the H2S-producing microorganismscounts at 15 °C (unwashed: y=0.5859x + 1.3119R2=0.8446; washed: y=0.8488x – 1.1493 R2=0.9874)and at 20 °C (unwashed: y=0.5765x+0.6180 R2=0.8734;washed: y=0.7625x–1.3103 R2=0.9468), lead to a betterexplanation of the sensory score measured by QIM. Thishigh correlation seems to indicate that, for whole gilt-head seabream stored in ice, the H2S-producing microor-ganisms are mainly responsible for sensory rejection ofthe fish.

Acknowledgments This study was supported by the EU FAIRCT 97–3127 (FAQUWEL) and the Spanish ALI99–1262-CE pro-jects.

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Table 2 Statistical analyses of H2S-producing microoganisms cultured in different conditions

Culture conditions Lots Days of storage

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Different letters in the same column indicate significant differences among culture conditions and different numbers in the same row in-dicate significant differences during the storage time (p≤0.05)

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