Sensory, Chemical, and Microbiological Attributes of Sea Bream (Sparus Aurata): Effect of Washing and Ice Storage

Abstract

The effect of washing and refrigeration on the quality of sea bream (Sparus aurata) was studied by monitoring the microbiological, chemical, and sensory changes of washed samples with tap water and non-washed samples. Washing dramatically reduced populations of bacteria, namely aerobic mesophilic bacteria (6.4–6.2 log cfu/g for gutted sea bream and 6.6–6.1 log cfu/g for whole gutted sea bream), psychrotrophic bacteria (6.7–6.0 log cfu/g and 6.8–6.1 log cfu/g), and H₂S-producing bacteria (6.4–5.4 log cfu/g and 6.7–5.5 log cfu/g) after 13 days of storage. Total volatile basic nitrogen (TVB-N) values showed no significant increase for all group of sea bream samples during storage. Of the chemical indicators of spoilage, trimethylamine (TMA) values of non-washed and washed sea bream increased very slowly and reached a final value of 1.35–1.47 mg/100 g (gutted fish) and 1.36–1.38 mg/100 g (whole fish), respectively (day 13). Sensory evaluation showed a good correlation with bacterial populations. On the basis of overall acceptability scores (sensory evaluation) a shelf-life of 12 days (washed and non-washed samples) was obtained for gutted and whole sea bream.

Keywords:

• Washing
• Sensory evaluation
• Chemical and microbiological quality
• Sea bream

INTRODUCTION

Fish is known to be a high protein and low fat meat.[1,2] Fish is one of the most highly perishable food products. During handling and storage, quality deterioration of fresh fish rapidly occurs and limits the shelf life of the product. Shelf life is defined as the period of time, under defined conditions of storage, for which a food product remains safe and fit for consumption. In other words, during this period, it should retain its desired sensory, chemical, physical, functional, or microbiological characteristics. The quality of fish degrades due to a complex process in which physical, chemical, and microbiological forms of deterioration are implicated. Enzymatic and chemical reactions are usually responsible for the initial loss of freshness, whereas microbial activity is responsible for the obvious spoilage and thereby establishes product shelf life.[3,4,5,6] In fact, the hygienic quality of fish and fishery products rapidly declines because of a cross-contamination from various sources. Bacterial spoilage of saltwater fish is caused by several Gram-negative bacteria; in particular, Shewanella putrefaciens was found to be very important for spoilage of packed cod, as well as Photobacterium phosphoreum, Pseudomonas spp., Achromobacter spp., Acinetobacter spp., Flavobacterium spp., and Aeromonas spp.[7,8] The application of low-dose irradiation, microwave cooking, high power ultrasound waves, by the addition of chemical preservatives, salting, curing has been previously reported to extend the shelf life of sea foods.[9,10,11,12,13,14,15] Washing has been employed for decontamination of fresh fish and then to prolong the storage period of fresh fish. Washing 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 (fruits, vegetables, and fish).[16] Pouring sea water or tap water on fish that are on the sales stands is a common application throughout the world. In some countries like Portugal it is an obligation to do so. In European countries, sea water is filtered and processed with ultra-violet light and then chlorinated (1 mg/l) before it is released to the water pipe system for the fish stands.[16] This type of water is used to decrease the bacterial load and inhibit bacterial growth without the development of unwanted taste and odour caused by the chlorine in fish. In our country, seawater or tap water is applied directly. It is not a legitimate obligation, but it is common and the aim is to keep the fish presentable and clean. Washing, in addition, is an application which is used to decrease the microbial flora. Fish as whole or gutted are ready for sale at the fish official markets after washing and stored in ice. The aim of this article was to determine the effects of washing with tap water on the quality of iced whole and gutted sea bream using sensory, chemical, and microbiological assessments.

MATERIALS AND METHODS

Fish Samples and Storage Conditions

Aqua-cultured fresh sea bream (Sparus aurata) of total 20 kg were cultivated in net cages in a Turkish fish farm and harvested during the period of June 2004. The fish were killed by immersion in ice slurry (hypothermia) and delivered to the laboratory as whole within 12 h of harvesting, packed in separate insulated polystyrene boxes with ice. The mean and standard deviations of the weight and length of the fish studied were 260 g (± 30) and cm 26.2 cm (± 0.6). Fish was washed once, after landing with running tap water. The fish were divided into four lots. Gutted control (AC), gutted washed (AW), whole control (BC), and whole-washed (BW). Gutting was carried out in the fish processing plant manually. In the washed lot, the fish was daily washed with tap water, and then stored again in the same conditions as the unwashed lot. During storage, ice was replenished when necessary. Boxes had perforated bottoms to allow drainage of melted crushed ice. Boxed fish were stored at 4°C.

Sensory Evaluation

Five experienced panellists analyzed all fish during days 1, 3, 5, 7, 9, 11, and 13 according to EU fish sensory schema (Table 1) for white fish.[17] The evaluation depends on the table created because white fish concept include haddock, cod, saithe, pollack, redfish, whiting, ling, hake, bream, anglerfish, pouting and poor cod, bogue, picarel, conger, gurnard, mullet, plaice, megrim, sole, dab, lemon sole, flounder, and scabbard fish. For the EU scheme the mean points of each fish were calculated and the fish were classified according to the following the quality bands: E<1, 1≤A<2, 2 ≤B<2.7, and 2.7≥ C points.[18]

Table 1 EU fish sensory scheme

Criteria	Extra Category	A Category	B Category	C Category (Not admitted)
Skin	Bright, iridescent pigment or opalescent; no discoloration	Pigmentation bright but not lustrous	Pigmentation in theprocess of becoming discolored and dull	Dull pigmentation
Skin mucus	Aqueous, transparent	Slightly cloudy	Milky	Yellowish grey, opaque mucus
Eye	Convex (bulging); black, bright pupil; transparent cornea	Convex and slightly sunken; black dull pupil; slightly opalescent cornea	Flat; opalescent cornea; opaque pupil	Concave in the centre; grey pupil; milky cornea
Gills	Bright color; no mucus	Less colored; transparent mucus	Brown/grey becoming discolored; thick, opaque mucus	Yellowish; milky mucus
Peritoneum (in gutted fish)	Smooth; bright; difficult to detach from flesh	Slightly dull; can be detached from flesh	Speckled; comes away easily from flesh	Does not stick
Smell of gills andabdominal cavity	Sea weedy	No smell of seaweed; neutral smell	Fermented; slightly sour	Sour
Flesh	Firm and elastic; smooth surface	Less elastic	Slightly soft (flaccid), less elastic; waxy and dull surface	Soft; scales easily detached from skin, surface rather wrinkled

Cooked Fish

The cooked fish were assessed using the simplified Torry Sensory Scheme for cooked white fish.[19] Panellists were asked to score odours, taste, and texture of fish using a 0–10 acceptability scale. Scale 10–9 is excellent, 8–7 is very good, 6–5 is acceptable, and <4 is unacceptable.

pH

Aliquots of 10 sardines were filleted from each sampling lot and homogenized using a food processor. Samples were prepared according to Vyncke,[20] by blending 10 g of the homogenate with 100 ml distilled water for 1 min at room temperature in a Ultra-Turrax (IKA T 25 Basic, Staufen, Germany). pH was monitored using a WTW-pH Meter (inoLab pH Level 1 model, Weilheim, Germany). Determinations were carried out in triplicate at days 1, 3, 5, 7, 9, 11, and 13 during the storage period.

Determination of Total Volatile Basic Nitrogen (TVB-N)

Total volatile basic nitrogen was determined according to the Antonacopoulos and Vyncke[21] method. For total volatile basic nitrogen (TVB-N), fish muscle (10 g) was homogenized with 6% perchloric acid (90 ml) for 1 min in an Ultra-Turrax. The homogenates were filtered through a filter paper (Whatman no. 1) and filtrates alkalized by 20% NaOH before distillation duplicate filtrates were distilled in a Velp Marka (Model UDK 140, Milan, İtaly) apparatus. The distillate titrated with 0.01 N HCl.

Determination of trimethylamin nitrogen (TMA-N)

This was determined by the method of AOAC.[22] Ten gram homogenized samples were weighed, blended with 90 ml of 7.5% trichloracetic acid solution and filtrated. Blended solution was fixed with formaldehyde (20%). Four ml extract was transferred into test tubes and 1 ml formaldehyde, 10 ml anhydrous toluene, and 3 ml K₂CO₃ solution were added. The tubes were shaken and 5 ml toluene layer was taken out by pipet. Five ml picric acid working solution (0.02%) was added. The contents were mixed and transferred to a spectrophotometric cell. Absorbance at 410 nm against the blank was measured. At the same time, standards were prepared and measured.

Microbiological Analyses

Three fish of each batch were randomly chosen. An area of approximately 10 cm² of the anterior-dorsal region and abdominal cavity of the right side of the fish was swabbed with a sterile swab, previously wetted with sterile peptone water (Merck, Kat No: 107228). Appropriate series of decimal dilutions were performed, from which surface inoculation was made using the 10 μl drop method in four solid medium. The media and incubations were: for aerobic plate count PCA agar (PCA, Merck, Kat No: 105463), 37°C, 1–2 days; for psychrotrophic bacteria PCA agar (PCA, Merck, Kat No: 105463), 7°C;[23] for H₂S-producing bacteria Iron agar 25°C, 2–3 days.[24] Counts were performed in duplicate and expressed as log cfu/cm².

Statistical Analysis

Significant differences between the samples were calculated by Excel XP 2003 by one –way analysis of variance (ANOVA) using a significance level of p < 0.05 by Tukey's Honestly Significant Difference test. A time dependent linear regression analysis was performed for the results obtained for sensory and microbiological analyses. Sperman's (r_s) (rank) and Pearson correlation (r) coefficients were used to compare physical, chemical and microbiological measurements to sensory panel results.[25]

RESULTS AND DISCUSSION

Sensory Analysis

In Figure 1 it can be seen that excellent and very good grades (E and A) were scored during the first 9 days storage for unwashed and washed fish. Moderate grades (B) were obtained between days 11 and 13 of storage for all groups. Unfit for sale of raw fish samples (C) were obtained 13 days of storage for unwashed and washed fish raw sea bream samples. Sensory results of washed and unwashed sea bream showed that day 9 was the beginning of spoilage. The samples were unfit for human consumption after 12 days. In ice with tap water washing samples, appearances of the gills, eyes, and mucus were better than in unwashed samples during the storage. Samples ice stored combined with tap water, had lower levels of intensive smell of gills and abdominal cavity than the gutted-unwashed samples after 9 days of storage. The application of washing with tap water decreased the formation of mucus and off-odour. Acceptability scores of all groups decreased significantly (p < 0.05) throughout storage (Figure 2). The samples had “very good” quality up to 9 days and “good” quality up to 11 days. The samples were unfit for human consumption at 12 days. The changes in sensory data were found to be insignificant (P > 0.05) in washed and unwashed sea bream. The storage life of whole, iced sea bream evaluated by sensory assessment of the raw fish has been reported to be 15–17 days.[26,27] The shelf life of whole ungutted sea bream stored in ice was found to be 17–18 days at 2 ± 2°C.[28] Lougovois et al.[29] concluded that shelf life of whole sea bream in ice is extremely limited, typical 15 days after catch. Poli et al.[30] reported that the limit of acceptability for cooked sea bass was 10 days. Erkan and Ozden[31] reported that the limit of acceptability for gutted and ungutted sea bass was 11 days. Huidobro et al.[32] found that the sensory evaluation scores of the cooked fillets (sea bream) had high scores for flavour, odour at the beginning of the storage period, but its score decreased at the end—unwashed samples (15 days) and washed samples (17 days). These findings are in agreement with the results described by Inácio et al.[16] They reported the demerit point of washed fish was lower than unwashed fish samples during the storage in ice.

Figure 1 Changes in EU score of aqua-cultured sea bream unwashed and washed with tap water.

Figure 2 Changes in cooked fish score of aqua-cultured sea bream unwashed and washed with tap water.

Chemical Analysis

The changes in pH, TVB-N, and TMA-N for unwashed and washed sea bream during the 13-day storage period in ice are shown in the Figures 3, 4, and 5. The values of pH ranged from 6.36 to 6.60 for AC groups, from 6.39 to 6.63 for AW, and from 6.55 to 6.67 for BC, from 6.39 to 6.75 for BW groups, respectively, during the 13-day storage period. Values of pH showed statistically significant (p < 0.05) changes for washed and unwashed (whole and gutted) sea bream samples during the entire period of storage. The pH was according to literature about 6.0–6.5 for fresh fish, and it increases during storage. The limit of acceptability is usually 6.8–7.0.[33] The pH increases are in agreement with the findings of Kyrana et al.;[27] Kyrana und Lougovois,[34]; Masyinom et al.;[35] Tejada and Huidobro;[36] Papadopoulus et al.;[37] Grigorakis et al.;[38] Taliadourou et al.[39] for sea bream species stored in ice.

Figure 3 Changes in pH level of aqua-cultured sea bream unwashed and washed with tap water.

Figure 4 Changes in TVB-N level of aqua-cultured sea bream unwashed and washed with tap water.

Figure 5 Changes in TMA-N level of aqua-cultured sea bream unwashed and washed with tap water.

The TVB-N content ranged from 17 mg/100 g to 15 mg/100 g flesh for unwashed sea bream groups and from 17 mg/100 g for washed samples groups, respectively, during the 13-day period of storage in ice. TVB-N values showed no significant increase for unwashed and washed sea bream during storage. TVB-N values showed significant fluctuation for all fish samples as a function of storage period indicating that TVB-N is a poor indicator of fish freshness, as also proposed by.[27,40] Similar TVB-N values were reported for whole fish[34,40] as well as for gutted fish..[40,41] For these species fish (Sparidae) a value of 25 mg/100 g has been designated as the limit acceptability.[42]

Microorganisms' activities are also related to chemical compounds (TVB, TMA, etc.), which are suggested as indicators of fish quality.[43] TMA is formed from bacterial use of TMAO, naturally occurring osmoregulating substance found in most marine fish species.[44] The quantity and presence of this compound depends on the species, size, sex, station of year, and so on.[45] Meyer[46] reported that H₂S- producing bacteria were both an important spoiler and TMAO reducer. Initial TMA-N values of unwashed and washed sea bream (groups AC-BC and AW-BW) samples were 0.31, 0.39–0.35, 0.36 mg/100 g, respectively. A content of 1.35, 1.76 mg/100 g was reached for the AC-BC samples after 13 days of storage whereas the AW-BW samples reached this content 1.47, 1.38 mg/100 g after 13 days of ice storage, respectively. Values of TMA-N showed not significant (p > 0.05) between washed and unwashed sea bream in both series during the entire period of storage. The concentrations of TMA-N in numerous fatty fish never reached the limit of 5 mg TMA-N/100 g, although the rejection limit in fish flesh is usually 5–10 mg TMA-N/100 g.[47] The level of TMA was typically around 0.07 mg/100 g in ice stored whole fish (sea bass) rejected by sensory panels.[37] Dalgaard et al.[48] reported that a population of 10⁸–10⁹ cfu/g of S. putrefaciens was considered crucial for TMA production. H₂S- producing bacteria (including S. putrefaciens) in this study reached at the end storage 5.04 log cfu/cm² (AC), 4.4 log cfu/cm² (AW), 5.7 log cfu/cm² (BC), 4.5 log cfu/cm² (BW) for sea bream samples, respectively. A highly significant correlation was found between TMA-N and H₂S- producing bacteria counts (AC, r = 0.841; AW, r = 0.779; BC, r = 0.753; BW, r = 0.874). Thus, TMA is not is not a particularly useful indicator of sea bream freshness. Similarly, low TMA values have been reported for whole fresh sea bass, trout, and sea bream.[27,36,49]

Table 2 Correlation coefficients between sensory score and microbial counts in whole and gutted sea bass

	AC	AW	BC	BW
Psychrotrophic bacteria	rs = 0.9062∗	rs = 0.9690∗	rs = 0.9334∗	rs = 0.9045∗
	rs = −0.9305∗∗	rs = −0.8844∗∗	rs = −0.9030∗∗	rs = −0.9121∗∗
Mesophilic bacteria	rs = 0.9917∗	rs = 0.9123∗	rs = 0.8940∗	rs = 0.9347∗
	rs = −0.8820∗∗	rs = −0.9361∗∗	rs = −0.8948∗∗	rs = −0.9494∗∗
H2S-producing bacteria	rs = 0.9055∗	rs = 0.9527∗	rs = 0.9188∗	rs = 0.8952∗
	rs = −0.9769∗∗	rs = −0.9437∗∗	rs = −0.9729∗∗	rs = −0.9335∗∗
∗Raw fish; ∗∗cooked fish.

Microbiological Analysis

Bacterial spoilage in refrigerated fish and fish products under aerobic storage conditions is caused by Gram-negative psychrotrophic organisms such as Pseudomonas, Alteromonas, Flavobacterium spp., and H₂S-producing bacteria (including Shewanella putrefaciens).[50] These bacteria have been reported to be the specific spoilage bacteria in fish from temperate and tropical waters and in fresh Mediterranean fish stored aerobically under refrigeration or ice storage.[4] Microbiological results are shown in Figures 6, 7, and 8. The sea bream initial mesophilic aerobic bacteria counts of 3.6, 3.4, 3.6, and 3.5 log cfu/cm² AC, AW, BC, and BW groups, respectively. These bacteria counts reached 6.1 log cfu/cm² (in AC) 5 log cfu/cm² (AW) (p < 0.05 significant), 5.7 log cfu/cm² (BC and BW) (p > 0.05, not significant), respectively after 11 days of storage. The evaluation of the mesophilic aerobic bacteria count was highly linear throughout storage in all groups [AC, R² = 0.955, y = 0.2x+2.10; AW,. Psychrotrophic bacteria counts for all groups increased with increasing time of ice storage (p < 0.05), (AC, R² = 0.79, y = 0.28x+1.60; AW, R² = 0.9151, y = 0.27x+1.64; BC, R² = 0.9376, y = 0.26x+1.60; BW, R² = 0.7939, y = 0.31x+1.36]. Initial psychrotrophic bacteria counts for AC, AW, BC, and BW groups were 3.8–3.5 log cfu/cm², respectively. Psychrotrophic bacteria counts reached 6.7–6.0 log cfu/cm² for AC and AW groups, and 6.8–6.1 log cfu/cm² for BC and BW groups' sea bream, respectively, after 13 days of storage. Initial H₂S-producing bacteria counts were 3.3 log cfu/cm² for all group fish sampling periods, respectively, with final counts of 6.4 log cfu/cm² (AC), 5.4 log cfu/cm² (AW)_, 6.7 log cfu/g (BC) and 5.5 log cfu/cm² (BW). The evaluation of the H₂S-producing bacteria counts was highly linear throughout storage in all groups (AC, R² = 0.8226, y = 0.26x+1.4; AW, R² = 0.8127, y = 0.19x+1.76; BC, R² = 0.8681, y = 0.30x+1.38; BW, R² = 0.8633, y = 0.18x+1.74). Statistical analysis showed significant differences were found only for the mesophilic aerobic, psychrotrophic, and H₂S-producing bacteria counts results in between the unwashed and washed groups (p < 0.05). (R² = 0.882, y = 0.23x+1.83; BC, R² = 0.860, y = 0.28x+1.71; BW, R² = 0.874, y = 0.23x+1.83). Positive and significant correlations between the increase of bacterial counts and the sensory score were detected only in the all sea bream samples. Zhuang et al.[51] have accepted the aerobic plate count limit of spoilage as 10⁶–10⁷ cfu/g. For fresh and refrigerated squid establish the microbial limit of acceptability at 6 cfu/g for psychrotropic bacteria.[52] Huidobro et al.[32] reported that mesophilic aerobic counts showed a slight increase for unwashed sea bream during storage, reaching a value of >7 log cfu/g (day 15), whereas for washing sea bream a corresponding value of >7 log cfu/g was recorded (day 17). Inacio et al.[16] found that mesophilic aerobic bacteria counts was >5 log cfu/cm² at the beginning of the storage period and its counts increased to over >7 log cfu/cm² and <7 cfu/cm² at the end of unwashed and washed whole scads (Trachurus trachurus) (12 days). They found initial H₂S-producing bacteria counts of 4 log cfu/cm² - 2 log cfu/cm² in case of unwashed and washed samples, respectively. They also stated that the H₂S-producing bacteria counts increased to counts of > 5 log cfu/cm² and 5 log cfu/cm² unwashed and washed fish days in ice. These findings are in agreement with the results described by Inacio et al.[16] and Huidobro et al.[32] Kyrana und Lougovois[34] reported for sea bass after 1-day ice storage mesophilic counts of 3 log cfu/g, H₂S-producing bacteria counts of 1.5 log cfu/g muscle with a maximum of 7 log cfu/g mesophilic count, 5 log cfu/g H₂S-producing bacteria counts after 14–15 day (rejection time). Taliadourou et al.[39] found initial of 3.5 log cfu/g in whole ungutted sea bass, respectively. They also stated that the total mesophilic counts increased to value of 5.5 log cfu/g in 9 days at 2 ± 2°C. These authors reported H₂S-producing bacteria (including S putrefaciens) of 6 log cfu/g 13 days of storage of whole ungutted sea bass in ice. Lougovois et al.[29] reported that the amount of mesophilic count and H₂S-producing bacteria in sea bream flesh at the time of rejection (15 days) in ice was 6.3 log cfu/g and 5 log cfu/g. Paleologos et al.[53] found a similar of mesophilic count and H₂S-producing bacteria count (6 log cfu/g and 5.8 log cfu/g) in ungutted sea bass muscle and, in gutted sea bass muscle 7.5 log cfu/g- 6.6 log cfu/g in ice storage day 11.

Figure 6 Changes in mesophilic counts of aqua-cultured sea bream unwashed and washed with tap water.

Figure 7 Changes in psychrotrophic bacterial counts of aqua-cultured sea bream unwashed and washed with tap water.

Figure 8 Changes in H₂S- producing bacteria of aqua-cultured sea bream unwashed and washed with tap water.

CONCLUSIONS

The effects of washing with tap water on microbiological, chemical, and sensory properties of sea bream (Sparus aurata) stored in cold storage (+4°C) were studied. Sea bream stored in ice as sensory, chemical, and microbiological results for washed and unwashed sea bream showed that day 9 was the beginning of spoilage. The samples were unfit for human consumption after 12 days. In this study, it was found the microbiological count of washed sea bream samples lower (p < 0.05) than unwashed samples during the storage. Ice storage combined with tap water washing was also found to have the most inhibiting effects on microbial count during the storage.

ACKNOWLEDGMENTS

This study was supported by the Research Fund of the University of Istanbul (Project No: UDP-591/12072005).

Notes

17. EC Regulation. Council Regulation (EC) N 2406/96 of 26 November 1996 Laying Down Common Marketing Standards for Certain Fishery Products. (OJ L 334, 23.12.1996) 1996; 1–15

22. Hungerford, J.M., Ed. AOAC Official Method 971.14 Trimethylamine Nitrogen in Seafood Colorimetric Method. Fish and Other Marine Products. In Official Methods of Analysis of AOAC International; Cunniff, P.; Ed.; 1998; 7.

42. 95/149/EG Entscheidung der Kommission vom 8. März 1995 über TVB- Grenzewerte für bestimmte Kategorien von Fischereierzeugnissen und die anzuwenden Analysemethoden. Amtsblatt nr. L 097 vom 29/04/1995, S.0084–0087.