EFFECTS OF OZONIZED FLAKE ICE ON SENSORY AND MICROBIOLOGICAL QUALITY OF PAGELLUS ERYTHRINUS

Abstract

To investigate the efficacy of ozone combined with both water and ice on the quality loss of Pagellus erithrynus during a 16-day chilling period, a total of 72 samples were collected and analysed. Sensory and microbiological analyses (skin and muscle) were carried out after 0, 3, 5, 7, 10, 12, 14 and 16 days. Collected samples were stored under flake ice (control batch), pre-treated with ozonized water (water ozone batch) and stored under ozonized flake ice (ozone batch). The highest freshness category up to 5 days, and the best antimicrobial success (lower than 5 Log CFU per cm2/g up to 16 days) were found in the ozone batch. The use of ozonized flake ice might represent a useful tool to enhance the shelf-life of fresh Pagellus erithrynus.

Headnote

ABSTRACT

To investigate the efficacy of ozone combined with both water and ice on the quality loss of Pagellus erithrynus during a 16-day chilling period, a total of 72 samples were collected and analysed. Sensory and microbiological analyses (skin and muscle) were carried out after 0, 3, 5, 7, 10, 12, 14 and 16 days. Collected samples were stored under flake ice (control batch), pre-treated with ozonized water (water ozone batch) and stored under ozonized flake ice (ozone batch). The highest freshness category up to 5 days and the best antimicrobial success (lower than 5 Log CFU per cm^sup 2^/g up to 16 days) were found in the ozone batch. The use of ozonized flake ice might represent a useful tool to enhance the shelf-life of fresh Pagellus erithrynus.

- Keywords: ozone, Pagellus erithrynus, shelf-life -

INTRODUCTION

Microorganisms have a critical rule in marine fish spoilage limiting the shelf-life of fresh fish. As well known, bacterial activity has a deep impact on sensory features (KOBATAKE et aL, 1992; ASAKAWA et aL, 1998; GENNARI et aL, 1999) and leads to a degradation of extractive nitrogen, amino acids, fats and sugars, and to a develop of ammonia-like off-flavors (GRAM et al., 2002) due to the presence of chemical molecules, such as trimethylamine (TMA), ammonia and hydrogen sulphide. The nitrogen substances are then decomposed by proteolytic bacteria (Achromobacter, Pseudomonas, Micrococcus, Bacillus, Alteromonas putrefaciens) until the liberation of amino acids (LISTON, 1980). The subsequent demolition of the substrate amino acid leads to the appearance of foul smelling volatile compounds, including amines, ammonia, short chain fatty acids, mercaptans and hydrogen sulfide (MAKARIOS-LAHAM and LEE, 1993). Spoilage is also linked to several factors like handling technique (unhygienic handling), fish species (moisture and fat content), storage condition (OLAESDÖTOR et aL, 1997) and starts immediately after the caught of fish (ABABOUCH et aL, 1991). Thus, reducing the growth of many spoilage microorganisms would enhance the quality of fishery products and subsequently increase their shelf-life. Several authors have published their findings on refrigeration systems based on the use of ozone to inhibit spoilage and preserve freshness. Different fish species have been examined after pre-treatment with ozonized water (KOTTERS et al., 1997) and ozonized slurry ice (CAMPOS etaL, 2005; AUBOURG et aL, 2009; ALVAREZ et aL, 2009). Ozone (03) has a strong oxidizing effect (lower only to fluorine) on a broad antimicrobial, antiviral and antifungal spectrum (GUZEL-SEYDIMA et aL, 2004). 03 has already been recognized as a valid method GRAS (Generally Recognized As Safe) and it can be used as antimicrobial agent in both aqueous and gaseous phase in the treatment, storage and processing of food including beef and poultry (USDA, 2002).

The aim of the present study was to investigate the efficacy of ozone combined with both water and flake ice on the quality loss of Pagellus erythrinus (or common pandora fish) during chilled storage; this fish is a commercially appreciated species of Sparidae family from the Mediterranean and the Black Sea, and the eastern coast of the Atlantic Ocean (Angola to Norway) (FISCHER et al., 1987). Sensory and microbiological analyses were carried out to evaluate the quality changes during a 16-day shelf life period.

MATERIALS AND METHODS

A total of 72 samples (Pagellus erithrynus) were collected at the local fishery market three hours after the caught neither headed nor gutted. Collected samples were divided in three batches, one of which used as control (CB, control batch). In the CB, chilling was guaranteed by covering fishery products with a thin plastic film with flake ice on the top. The second batch (WOB, water ozone batch) was firstly washed with ozonized water (3 mg/L) for 3 min and then chilled by keeping samples under a thin plastic film with flake ice on the top. In the last batch (OB, ozone batch) chilling was done by covering samples with ozonized ice (3mg/L). Treated water and ice were obtained by injection of ozone using a prototype (OXITECH S.r.l., Italy). In both cases (ozonized ice and flake ice), the fish/ice ratio was 1:1. During the experimental time the ice was renewed repeatiy. On fixed days (0, 3, 5, 7, 10, 12, 14 and 16), three samples per each batch were taken and transported cooled to the laboratory where sensory and bacteriological analyses were carried out within 3 hours. Sensory analyses were done by a panel of five untrained panelists up to sixteen days. A quality index classifying samples in freshness categories, highest quality (E) to unacceptable (C), was attributed to each sample in accordance with parameters listed in Table 1 (Council Regulation 2406/96/ EC). To evaluate the ozone efficiency on the microbiological contamination of fishery products two aliquots were collected from each sample (skin surface and muscle). The skin surface was processed by using the double wet/dry swabbing technique over a 5 cm2 area delimited by a sterile template. Briefly, the wet swab was rubbed vertically, horizontally, then diagonally across the template surface (20 sec). Swabbing was then repeated with a dry swab. Swabs were placed into a sterile stomacher bag and homogenized in 10 mL of 0.1% peptone water (Oxoid Ltd., Hampshire, UK) for 60 sec. The second aliquot (5 g of muscles) was aseptically cut off using a sterile blade and then placed in a sterile stomacher bag. Muscles were homogenized in 45 mL of 0.1% peptone water (Oxoid Ltd.) for 60 sec. Microbiological analyses were done by culture after a dilution step. For enumeration of total bacterial count a subset (0.1 mL) from each dilution was inoculated onto Plate Count Agar (Oxoid Ltd.) and incubated at 30°C for 3 days (TBC 30°C) and at 5°C for 10 days (TBC 5°C). Enumeration of proteolytic bacteria was estimated by plating 0.1 mL from each dilution onto casein-agar medium (PHAFF et aL, 1994), as described by BEN-GIGIREY et aL (2000). Microbiological counts were expressed as Log CFU per cm2/g of the average values of three independent determinations. One-way ANOVA with Tukey post tests was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA. A confidence interval at the 95% level (P < 0.05) was considered to explore significance of differences among microbiological parameters throughout storage for each refrigeration system.

RESULTS AND DISCUSSION

Results of sensory analyses are reported in Table 2. A score decrease was observed gradually. The appearances of skin mucus and eyes limited firstly the fish acceptability in all batches. CB showed good quality until day 3 (E and A categories) and acceptable until day 10. WOB retained a good quality until day 5 and acceptable until day 14. The best results were found in OB, that showed good quality until day 7 and acceptable until day 16 highlighting the effectiveness of ozonized flake ice to keep freshness. Sensory results in this study are in agreement with previous studies on the application of ozone to extend the shelf life of different fish species as rockfish (KOETTERS et al., 1997), catfish fillets (KIM et al., 2000) and Pagellus bogaraveo where the highest sensory quality was assessed up to 9 days after treatment with flow ozonized ice (ALVAREZ et al., 2009). Microbiological results regarding skin aliquots are reported in Table 3. Regarding TBC 30°C, statistically significant differences (P < 0.05) were evidenced on day 5 until day 16 between OB and CB. On the contrary, differences were not evi- denced between WOB and CB. The average difference determined for CB and OB up day 16 was 0,56 Log units. In OB the microbial count was below 2 Log CFU/cm2 up to 10 day and did not reach concentrations of 4 Log CFU/cm2 after 16 days of storage. The microbial load in all the batches was below 6 Log CFU/cm2 up to 16 days, value considered necessary to induce fish spoilage (GRAM and HUSS, 1996). With regard to TBC 5°C, statistically significant differences (P < 0.05) were evinced between OB and CB starting from day 3 up to 16 (day 12 excepted). Significant differences were not proved between WOB and CB. In both batches microbial load was 2 Log CFU/cm2 at day 3 with a significant increase at day 10. The average difference between OB and CB was 0,59 Log units. Regarding proteolytic bacteria statistically significant differences (P < 0.05) were evinced between OB and CB at days 5,7,14,16. Differences between WOB and CB were not observed. The average difference between OB and CB was 0,55 Log units. With regard to muscle aliquots, comparative results are reported in Table 4. In TBC 30°C significant differences were found between CB and OB from day 7 to the end, but no differences were found between WOB and CB samples; the average difference between OB and CB was 0,41 Log units. In TBC 5°C significant differences (P < 0.05) were observed between CB and OB from day 3 to day 16 (day 5 excepted) and between WOB and CB at days 3,7 and 12. The average difference between CB and OB was 1,03 Log units and between WOB and CB was 0.23 Log units. Concerning proteolytic bacteria significant differences (P < 0.05) were evidenced between CB and OB at days 3, 5,7 and 14, but not evidenced between CB and WOB: the average difference between CB and OB was 0,40 Log units.

To the best of our knowledge, this is the first study providing evidence of the antimicrobial efficacy of ozonized flake ice. The batch treated with ozonized flake ice (OB) showed the highest freshness category (up to 5 days) and the best antimicrobial success. In agreement with recent studies (AUBOURG et al., 2006; AUBOURG et al., 2009; CAMPOS et al., 2005; LU et al., 2012) the ozone activity was confirmed by reducing fish spoilage bacteria both in muscle and skin aliquots during storing time. ALVAREZ et al. (2009) by using a combination of flow ice and ozone found an average reduction of 0.56, 0.46 and 0.46 Log units respectively for total aerobes and psychrotrophes and proteolytic bacteria in Pagellus bogavero muscle. On the other hand, in our study a lower reduction (0.41 and 0.4 Log units) for TBC 30 and 5°C and a grater reduction (1.03 Log units) for proteolytic was demonstrated. Based on our data, an ozone concentration of 3 mg/L might positively affect sensory quality and be effective in slowing down microbial activity when used in combination with flake ice. Differently, the use of the same concentration in water pre-treatment slightly influenced sensory features and did not significantly affect microbial contamination.

References

REFERENCES

Ababouch L.H., Afilal M.E., Benabdeljelil H. and Busta F.F. (1991): Quantitative changes in bacteria, amino acids and biogenic amines in sardine (Sardina pilchardus) stored at ambient temperature (25° to 28°C) and in ice. International Journal of Food Science & Technology 26: 297-306.

Alvarez V., Feàs X., Barros-Velôzquez J. and Aubourg S.P. (2009): Quality changes of farmed blackspot seabream (Pagellus bogaraveo) subjected to slaughtering and storage under flow ice and ozonised flow ice. International Journal of Food Science & Technology 44: 1561-1571.

Asakawa M., Sadakata Y., Araki T, Sumi T. and Nakagawa H. (1998): Purification and characterization of the alkaline serine protease produced by Bacillus sp. N4 strain from fish skin mucus. Fish Science 64: 793- 797.

Aubourg S.P, Testi S., Sanxuàs M., Gil C. and BarrosVelàzquez J. (2009). Improved quality and shelf life of farmed trout (Oncorhynchus mykiss) by whole processing in a combined ozonised flow ice refrigeration system. International Journal of Food Science & Technology 44: 1595-1601.

Aubourg S.P., Losada V., Gallardo J.M., Miranda J.M. and Barros-Velázquez J. (2006). On-board quality preservation of megrim (Lepidorhombus whiffiagonis) by a novel ozonised-slurry ice system. European Food Research and Technology 223: 232-237.

Ben-Gigfrey B., Vieites-Baptista de Sousa J.M., Villa T.G. and Barros-Velázquez, J. (2000). Characterization of biogenic amine-producing Stenotrophomonas maltophilia strains isolated from white muscle of fresh and frozen albacore tuna. International Journal of Food Microbiology 57: 19-31.

Campos C., Rodríguez Ó., Losada V., Aubourg S. and BarrosVelázquez J. (2005). Effects of storage in ozonised sluriy ice on the sensoiy and microbial quality of sardine (Sardina pilchardus). International Journal of Food Microbiology 103: 121-130.

COUNCIL REGULATION (EC) No. 2406 of 26 November 1996 laying down common marketing standards for certain fishery products

Fischer W., Schneider M. and Bauchot M.L. (Eds.) (1987). Fiches FAO d'identification des espèces pour les besoins de la pêche. (Révision 1). Méditerranée et mer Noire. Zone de pêche 37. Volume I. Végétaux et Invertébrés. Publication préparée par la FAO, résultat d'un accord entre la FAO et la Commission des Communautés Européenes (Project GCP/INT/442/EEC) financée conjointment par ces deux organisations. Rome, FAO, Vol. 1.

Gennari M., Tomaselli S. and Cotrôna V. (1999). The microflora of fresh and spoiled sardines (Sardina pilchardus) caught in Adriatic (Mediterranean) sea and stored in ice. Food Microbioly 16: 15-28.

Gram L. and Dalgaard P. (2002). Fish spoilage bacteria. Problems and solutions. Current Opinion in Biotechnology 13:262-266.

Gram L. and Huss H. 1996. Microbiological spoilage of fish and fish products. International Journal of Food Microbiology 33: 121-137.

Guzel-Seydim Z.B., Greene A.K. and Seydim A.C. (2004). Use of ozone in the food industry. Lebensmittel-Wissenschaft & Technologie 37:453-460.

KimT.J., Silva J.L., Chamul R.S. and ChenT.C. (2000). Influence of ozone, hydrogen peroxide or salt on microbial profile, TBARS and color of channel catfish fillets. Journal of Food Science 65: 1210-1213.

Kobatake M., Kreger van Rij N.J.W., Placido M.T.L.C., and van Uden N. (1992) : Isolation of proteolytic psychrotrophic yeasts from fresh raw seafoods. Letters in Applied Microbiology 14: 37-42.

Koetters J., PrahstA., SkuraB., Rosenthal H., BlackE.A. and Rodigues-Lopez J. (1997). Observationsand experiments on extending shelf-life of "rockfish" (Sebastes spp.) products with ozone. Journal of Applied Ichthyology 13: 1-8.

Liston J. (1980). Microbiology in fishery sciences. In: J.J. Connell (Ed.), Advances in fishery science and technology (pp. 138el57). Surrey: Fishing News Books.

Makarios-Laham I.K. and LeeT.C. (1993). Protein hydrolysis and quality deterioration of refrigerated and frozen seafood due to obligately psychrophilic bacteria. Journal of Food Science 58: 310-313.

Olafsdöttir G., Martinsdôttir E., Oehlenschläger J., Dalgaard P., Jensen B., Undeland I., MackieI., Henehan G., Nielsen J. and Nilsen H. (1997). Methods to evaluate fish freshness in research and industry. Trends in Food Science & Technology 8: 258-265.

Phaff H.J., Starmer W.T., Lachance M.A. and Ganter P.F. (1994). Candida caseinolytica sp. nov., a new species of yeast occurring in necrotic tissues of Opuntia and Stenocereus species in the Southwestern United States and Baja California, Mexico. Applied and Environmental Microbiology 44: 641- 645.

USDA final rule on ozone dated 12/17/2002, FSIS Directive 7120.1.

Paper recieved July 5, 2013 Accepted October 17, 2013