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International Journal of Food Microbiology 97 (2004) 209 – 214 www.elsevier.com/locate/ijfoodmicro Short communication Effects of modified atmosphere and vacuum packaging on microbiological and chemical properties of rainbow trout (Oncorynchus mykiss) fillets Xqkriye Arashisara, Olcay Hisara, Mqkerrem Kayab,*, Telat Yanika a b Agricultural Faculty Fisheries Department, Atatuărk University, Erzurum 25240, Turkey Agricultural Faculty Food Engineering Department, Atatuărk University, Erzurum 25240, Turkey Received 16 September 2003; received in revised form May 2004; accepted 26 May 2004 Abstract Microbial (psychrotrophic, mesophilic aerobic bacteria and Enterobacteriacae counts), and chemical analysis [pH, total volatile bases nitrogen (TVB-N), lipid oxidation (Thiobarbituric acid reactive substance, TBARS)] of rainbow trout (Oncorynchus mykiss) fillets in air (control), vacuum and modified atmosphere packaging (MAP) with various gas mixtures conditions at 4F1 8C were determined The gas mixtures evaluated were 100% CO2, 2.5% O2+7.5% N2+90% CO2 and 30% O2+30% N2+40% CO2 Psychrotrophic bacteria count was above 1Â107 cfu/g on the 12th day in 100% CO2 However; mesophilic bacteria count was below 1Â106 cfu/g at the end of the 14-day storage period Enterobacteriaceae count was significantly lower in samples packaged with MAP Lipid oxidation increased rapidly after days of storage in the samples containing 30% O2 While minimum TBARS values were recorded in fillets containing 100% CO2 and vacuumed fillets, the lowest TVB-N values were obtained in fillets with 100% CO2 D 2004 Published by Elsevier B.V Keywords: Rainbow trout; Modified atmosphere packaging; Specified spoilage level; Psychrotrophic bacteria; TBARS Introduction Fresh fish are highly perishable products due to their biological composition Spoilage of fish muscle results from changes brought about by biological * Corresponding author Agricultural Faculty Food Engineering Department, Atatqrk University, Erzurum 25240, Turkey Tel.: +90 442 231 22 04; fax: +90 442 236 09 58 E-mail address: mkaya@atauni.edu.tr (M Kaya) 0168-1605/$ - see front matter D 2004 Published by Elsevier B.V doi:10.1016/j.ijfoodmicro.2004.05.024 reactions such as oxidation of lipids, reactions due to activities of the fish’s own enzymes, and the metabolic activities of microorganisms These activities lead to a short shelf life in fish and other seafood products (Statham and Bremner, 1989; Ashie et al., 1996; Gram and Huss, 1996; Gobantes et al., 1998) MAP offers multiple advantages to the fish industry and the consumer Various atmospheres have been examined in fish packaging (Parkin et al., 1981; Barnett et al., 1987; Stammen et al., 1990; Farber, 210 SS Arashisar et al / International Journal of Food Microbiology 97 (2004) 209–214 1991; Reddy et al., 1994; Randell et al., 1995; Randell et al., 1997; Gimenez et al., 2002; Sivertsvik et al., 2002) Oxygen, nitrogen and carbon dioxide are the most usual gases used in MAP (Randell et al., 1997; Gimenez et al., 2002) Oxygen causes oxidative rancidity in fatty fish, stimulates growth of aerobic bacteria and inhibits growth of strictly anaerobic bacteria Nitrogen delays oxidative rancidity and inhibits the growth of aerobic microorganisms by displacing the oxygen in packs (Stammen et al., 1990; Farber, 1991; Philips, 1996; Church, 1998) Since, carbon dioxide acts as an antimicrobial agent (Stammen et al., 1990), it inhibits the growth of microorganisms during the logarithmic phase and extends the lag phase (Genigeorgis, 1985; Ohlsson, 1994; Church, 1994; Philips, 1996) Different concentration of CO2 has some effects on bacteria (Church, 1998) Statham (1984) explains that bweak acids are known to have antimicrobial activity in their undissociated form; therefore carbonic acid is unique as a microbial inhibitor since at pH values near neutrality at least one half of the acid is in the undissociated form The pK value for the first dissociation is 6.37 yielding hydrogen and bicarbonate ionsQ It was reported that high concentrations of CO2 for fish should be avoided since it dissolves into the fish juice and deforms the package (Stenstroăm, 1985) However, the use of a MAP with an enhanced carbon dioxide level has been also shown to extend the shelf life of fresh fishery products by retarding microbial growth (Farber, 1991; Reddy et al., 1994; De La Hoz et al., 2000; Emborg et al., 2002) Gas mixtures containing CO2 and O2 have also extended the shelf life (Stenstroăm, 1985; Lopez-Galvez et al 1995; Debevere and Boskou, 1996; Pastoriza et al 1996) It has been reported that 40% CO2 and 60% O2 significantly inhibited the microbial growth in cod Stenstroăm (1985), 35–100 CO2 and O2 or N2 mixtures in packed shrimp (Lannelongue et al., 1982) and 50– 100% CO2 also greatly inhibited the microbial growth in MAP compared to air in spotted shrimp (Layrisse and Matches 1984) It has been recommended that gas mixtures for the lean fish and fat fish should be 40% CO2+30% N2+30%O2 and 60% CO2+40% N2, respectively (Cann et al., 1983; Cann et al., 1984, Church, 1998) However, for the maximum microbial inhib- ition, 100% CO2 has been used, i.e in cod fillets Stenstroăm (1985) Considering this fact, the establishment of the gas mixtures related to the target product is very important Thus, the present study was undertaken to determine the effects of vacuum and MAP atmospheres with various gas mixtures, 100% CO2, 2.5% O2+7.5% N2+90% CO2 and 30% O2+30% N2+40% CO2, on microbiological (psychrotrophic, mesophilic aerobic bacteria and Enterobacteriaceae counts), and chemical changes [pH, total volatile bases nitrogen (TVB-N), lipid oxidation (TBARS)] of rainbow trout (Oncorynchus mykiss) fillets during storage at 4F1 8C Materials and methods 2.1 Preparation and storage of samples Fresh water rainbow trout with an average weight of 200 g reared in a farm, located in Research and Extension Center of Fisheries Department in Agricultural Faculty at Atatqrk University in Erzurum, were transferred to the Meat Processing Laboratory in Food Science Department, and decapitated and filleted by hand Two fillets were obtained from each fish by removing the head and bone of fish The fillets were divided into five groups and packaged in five different atmospheres by using a Multivac packaging machine (Multivac A 300/16, Sepp Haggenmuller, D 87787 Wolfertschwenden, Germany) Packaging material was a film bag with 15Â25 cm OPA/EVOH/PE (Oriented Polyamid-EVOH-Polyetilen, UPM-Kymmene Walki Films, Finland) and with low gas permeability (oxygen transmission rate of cm3/m2/ days atm at 23 8C, nitrogen transmission rate of cm3/m2/days atm at 23 8C, carbon dioxide transmission rate of 23 cm3/m2/days atm at 23 8C and water vapour transmission rate of 15 g/m2/days atm at 38 8C) The final gas/product ratio in all pouches was about 2:1 (v/w) for MAP conditions The composition of gas mixtures was adjusted as 100% CO2, 2.5% O2+7.5% N2+90% CO2 and 30% O2+30% N2+40% CO2 by a commercial company (Karbogaz, Istanbul, a partner of PRAXAIR, Danbury) in the present study The fillets were inserted in film bags and sealed after removal of air in vacuum packaging The fillets were placed in film bags and sealed without removal SS Arashisar et al / International Journal of Food Microbiology 97 (2004) 209–214 of air for the control group All of the fillets were stored at 4F1 8C for 14 days The fillets in duplicate were subjected to microbial, physical and chemical analyzes on the 0, 2, 4, 6, 8, 10, 12 and 14th days of the storage period 2.2 Microbial analysis Tissue samples were taken to determine total aerobic bacteria and Enterobacteriaceae counts from each of two different fish stored under five different storage conditions (air, vacuum, 100% CO2, 2.5% O2+7.5% N2+90% CO2 and 30% O2+30% N2+40% CO2) Each of 25 g fish muscle was removed aseptically and homogenized for in a Stomacher 400 (Lab Stomacher Blander 400-BA7021, Sewardmedical) bag containing 0.85% NaCl solution Further decimal dilutions were made and then 0.1 ml of each dilution was pipetted onto the surface of plate count agar (PCA, Merck) PCA plates were then incubated for days at 10 8C for psychrotorophic bacteria count and for days at 37 8C for mesophilic bacteria count Enterobacteriaceae was determined in Violet-RedBile-Glucose agar (VRBG-agar, Merck) plates incubated anaerobically at 30 8C for days All counts were expressed as log10 cfu/g McMeekin et al (1993) reported that usually a bspecified reactive levelQ should be used to show unacceptable levels in food products, therefore, a horizontal line was used in each figure to show these spoilage levels in the present study 2.3 Lipid oxidation Lipid oxidation, measured as Thiobarbituric acid reactive substances (TBARS) values, was determined according to Lemon (1975) 2.4 Total volatile base nitrogen (TVB-N) A vapour distillation method was used for total volatile bases nitrogen (TVB-N) estimation (Anonymous, 1988) The results were expressed as mg TVBN/100g 2.5 pH value The pH values were recorded by using a Schott model pH meter (Schott, Lab Star pH) after homog- 211 enization of each À10 g fish muscle sample in 100 ml distilled water 2.6 Statistical analysis Psychrotrophic, mesophilic aerobic bacteria, Enterobacteriacae counts, pH, total volatile bases nitrogen (TVB-N) and lipid oxidation (TBARS) of rainbow trout fillets in air (control), vacuum and modified atmosphere packaging (MAP) with various gas mixtures conditions at 4F1 8C were determined Data were checked for normal distributions with normality plots prior to one-way analysis of variance (ANOVA), and followed by Duncan’s multiple range test to determine significant differences among means at a=0.05 level (Duncan, 1971) Results and discussion 3.1 Microbiological analysis The changes in mesophilic and psychrotrophic bacteria counts throughout the storage of refrigerated rainbow trout fillets packaged in air, vacuum, 100% CO2, 2.5% O2+7.5% N2+90% CO2 and 30% O2+30% N2+40% CO2 are presented in Fig 1a and b Both mesophilic and psychrotrophic bacteria count in fillets under different atmosphere conditions increased with length of storage at 8C After days in air, packaging psychrotrophic and mesophilic bacteria counts reached above than 107 and 106 cfu/g, respectively However, growth of aerobic bacteria in the fillets packaged under 100% CO2 was slower during storage, psychrotrophic bacteria count reached 107 cfu/g at the 10th day and mesophilic bacteria count reached 105 cfu/g at 14th day of storage Effects of 90% CO2 and 40% CO2 on bacterial growth were lower than that of the 100% CO2 (Fig 1a and b) An explanation for this situation could be the high concentration of CO2 which has bacteriostatic and fungustatic properties It is known that antimicrobial effect of CO2 increases depending on the solubility which is increased by the low water temperature (Stammen et al., 1990; Ashie et al., 1996) and CO2 prolongs lag phase of bacterial growth and increases generation time (Philips, 1996) Delaying of bacterial growth in fish fillets packaged in MAP including 212 SS Arashisar et al / International Journal of Food Microbiology 97 (2004) 209–214 Fig Changes in psychrotrophic (a), mesophilic (b), Enterobacteriaceae (c) counts, TVB-N levels (d), TBARS levels (e) and pH values (f) on rainbow trout fillets storaged in different atmospheres at 8C Upper areas of horizontal lines are unacceptable in each figure various CO2 contents was also previously reported in some studies i.e in cod (Cann et al., 1983), in rainbow trout (Barnett et al., 1987; Randell et al., 1997; Gimenez et al., 2002), in sole (Lopez-Galvez et al., 1998) and in herring fillets (Randell et al., 1997) The effect of storage time x atmosphere interaction on Enterobacteriaceae count was important ( pb0.05) Enterobacteriaceae count was below 1Â104 cfu/g in fillets packaged under 100% CO2 and 90% CO2 on day It reached to 105–106 cfu/g in control and vacuum packaged samples in day of storage time MAP caused a significant decrease in Enterobacteriaceae count in fillets and the lowest average was obtained in fillets packaged under 100% CO2 Enterobacteriaceae counts in fillets packaged under 100% CO2 were approximately 0.5 and 1.5 log units lower in fillets packaged under 90% CO2 and 40% CO2, respectively (Fig 1c) Similarly, Stenstroăm (1985) found that CO2 concentration was an important factor in cod fillets stored at 8C in MAP and 100% CO2 inhibited Enterobacteriaceae more effec- tively Some other studies have also shown that concentrations of 20% and 40% CO2 inhibited Enterobacteriaceae family species in Sole fillets (Lopez-Galvez et al., 1998), in hake steaks (Ordonez et al., 2000) and in salmon steaks (De La Hoz et al., 2000) at 8C It has also been reported that CO2 delayed spoilage of fresh seafood by inhibiting psychrotrophic, aerobic and Gram-negative bacteria (Finne, 1982) 3.2 TVB-Nitrogen Storage timeÂatmosphere interaction had significant effects on TVB-N values (Fig 1d) TVB-N values were above 20 mg/100 g in all groups except in the group with 100% CO2 on day 10 These values reached above 25 mg/100 g in all groups except the groups with 100% CO2 and 90% CO2 on day 12 Finally, they reached above 35 mg/100 g in all samples at day 14 The findings of the present study suggested that 25 mg/100 g TVB-N level in tissue can SS Arashisar et al / International Journal of Food Microbiology 97 (2004) 209–214 be considered as the highest acceptable level for rainbow trout similarly to Gimenez et al (2002) 3.3 Lipid oxidation Oxidative rancidity may become a problem if higher than normal levels of oxygen are used (Finne, 1982) Stammen et al (1990) reported that rancidity due to oxidation of polyunsaturated fatty acids (PUFA) in some fish may be a problem in modified atmosphere with O2 No significant differences were observed between vacuum packaged and 100% CO2 group with respect to TBARS value The highest average TBARS value was obtained from fillets packaged with 30% O2 (Fig 1e) Similar results were reported by Pastoriza et al (1996) in hake slices packaged in MAP Gimenez et al (2002) reported that lipid oxidation was significantly higher in gas packages with 20% and 30% O2 than in those with 10% O2 in rainbow trout fillets There were fluctuations in TBARS values, the possible reason for that may be that the different modified atmospheres could produce different oxidation products (Raharjo et al., 1993; Doe et al., 1998) Storage timeÂatmosphere interaction had significant effects ( pb0.05) on TBARS values (Fig 1e) TBARS values increased rapidly after day in gas packages with 30% O2 Finne (1982) reported that higher TBA values were determined from 40% CO2+60% O2 and 70% CO2+30% O2 than other atmospheres (100% CO2; 70% CO2+30% N2 and 40% CO2+60% N2) in swordfish steaks packed under different gas atmospheres 3.4 pH Statham (1984) reported that pH values were slightly reduced in fish flesh with the dissociation of carbonic acid in general Storage timeÂatmosphere interaction did not affect significantly pH values (Fig 1f) No significant differences were observed among pH values determined from different atmosphere conditions Similarly Silva and White (1994) did not find significant differences between packing atmospheres (air, low CO2 and high CO2) Moreover, Barnett et al (1982) reported also no significant changes in pH values of salmon flesh reared in sea water stored at 90% CO2 However, the effect of 213 storage time on pH was significant ( pb0.05) The highest average pH (6.47) was obtained on day 12 (Fig 1f) All of the pH values except control group were in accordance with the findings of Gimenez et al (2002) 3.5 Conclusions Fig 1a–c shows clearly that bacterial growth is increasingly depressed by higher concentrations of CO2 Fig 1e shows that oxidation products are depressed by lower concentrations of oxygen Although 40% CO2+30% N2+30% O2 is recommended for lean fish, it has been found to be the worst choice for fresh water rainbow trout stored at 8C This is because of the increased oxygen It is not surprising that it is worse than air which has 20% oxygen If much of the CO2 becomes dissolved in the flesh the atmosphere surrounding the pack will have an even higher concentration of O2 and N2 than the original mixture References Anonymous, 1988 Unterschung von Lebensmitteln Bestimmung des Gehaltes von flqchtigen stickstoffhaltigen Basen (TVB-N) in Fischen und Fischerzeugnissen Referenzerfahren Amtliche Sammlung von Untersuchunsverfahren nach 35 LMBG, 80 Ashie, I.N.A., Smith, J.P., Simpson, B.K., 1996 Spoilage and shelflife extension of fresh fish and shellfish Critical Reviews in Food Science and Nutrition 36, 87 – 121 Barnett, P.J., Stone, F.E., Roberts, G.C., Hunter, P.J., Nelson, W., Kwok, J., 1982 A study in the use of a high concentration of carbon dioxide in a modified atmosphere to preserve salmon Marine Fisheries Review 44 (3), 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Jeksrud, W.K., Rosnes, M.J.T., 2002 A review of modified atmosphere packaging of fish and fishery products— significance of microbial growth, activities and safety International Journal of Food Science and Technology 37, 107 – 127 Stammen, K., Gerdes, D., Caporaso, F., 1990 Modified atmosphere packaging of seafood Food Sciences and Nutrition 29, 301 – 331 Statham, J.A., 1984 Modified atmosphere storage of fisheries products: the state of the art Food Technology in Australia 36 (5), 233 – 239 Statham, J.A., Bremner, H.A., 1989 Shelf-life extension of packaged seafoods—a summary of a research approach Food Australia 41 (2), 614 – 620 Stenstrfm, I.M., 1985 Microbial flora of cod fillets (Gadus morhua) stored at 8C in different mixtures of carbon dioxide and nitrogen/oxygen Journal of Food Protection 48, 585 – 589  ... Effect of modified atmosphere on shelf-life of iced fresh hake slices Journal of the Science of Food and Agriculture 71, 541 – 547 Philips, O.C.A., 1996 Review, modified atmosphere packaging and. .. enriched atmospheres Journal of the Science of Food and Agriculture 80, 1831 – 1840 Parkin, K.L., Wells, M.J., Brown, W.D., 1981 Modified atmosphere storage of rockfish fillets Journal of Food... 1982 Modified- and controlled -atmosphere storage of muscle foods Food Technology 36 (2), 128 – 133 Genigeorgis, C., 1985 Microbial and safety implications of the use of modified atmospheres to