Effect of salting and packaging on liquid-smoked rainbow trout fillets during refrigerated storage

The aim of this study was to investigate the effect of salting and packaging treatments on liquid-smoked rainbow trout fillets during refrigerated storage (4°C) for 120 days. Treatments included the following: V1 (vacuum packaged35% brine), M1 (modified atmosphere packaged50% CO2 + 50% N235% brine), V2 (vacuum packaged70% brine) and M2 (modified atmosphere packaged70% brine). Fillets were subjected to microbiological (total aerobic mesophilic bacteria, psychrotrophic bacteria, Pseudomonas, lactic acid bacteria, Enterobacteriaceae, yeast and mould) and chemical (pH, thiobarbituric acid reactive substances-TBARS, total volatile base nitrogen-TVB-N) analyses on 0, 15, 30, 45, 60, 75, 90, 105 and 120 th days. The number of microorganisms, TBARS and TVB-N values increased according to increment of storage. Modified atmosphere packaging (MAP) gave better results than vacuum packaging in terms of all investigated parameters. Moreover, M2 was effective in extending the shelf life of rainbow trout fillets.


INTRODUCTION
Microorganisms can reproduce in the muscle tissue of aquatic products because these are not only sensitive but also have rich nutrient content.So, the conservation and handling of aquatic products are very important (Babadoğan, 1998).One of the conservation processes of aquatic products is smoking (Bellagha et al., 2007).Smoking is one of the oldest methods of food presservation and widely used in fish processing (Muratore and Licciardella, 2005;Stolyhwo and Sikorski, 2005).
The shelf-life of smoked fish products depends largely on the intial bacterial contamination of the raw material; on the decrease in the water activity (aw) of the tissues due to brining and pre-drying; on the activation of putrefactive microflora due to the heat treatment; on the amount of smoke compenents that penetrate the product and on the temperature, air humidity and oxygen levels during storage (Sikorski et al.,1990;Ibrahim et al., 2008).
There are three methods used to smoke fish: the traditional method by combusition, at either low temperature (cold smoking ≤30°C) or high temperature (hot smoking ≥60°C); use of a high voltage electrostatic field which accelerates smoke deposition; and use of liquid smoked fish (Goulas and Kontominas, 2005).Liquid smoke flavorant is obtained from condensation of wood smoke and generally used as a flavoring agent (Siskos et al., 2005).Easy application, speed, product uniformity, low price and compatibility with the environment are the advantages of the use of smoke flavorings E-mail: pinaroguzhan@ardahan.edu.tr.Tel: +90 478 2115000.Fax: +90 478 2113275.
as compared to traditional smoking techniques (Simon et al., 2005;Alçiçek, 2011).Salting process is itself a preservation technique, is used as a preliminary operation for many processing technology (smoking, drying and marinating processes).The first step of the fish smoking process is salting.The main purpose of salting the fish meat is a part of the elimination of water.Bacterial activity is largely prevented in high salt concentrations.High salt concentration prevents bacterial activity which can generate spoilage in fish.Consequently, salting process, significantly increase the shelf-life of fish (Ismail and Wootton, 1992).
Beyong the prevention of microbial growth and reduction of water activity, NaCl is an essential ingredient in processed meat products for its contribution to the water-holding capacity, facilitating the solubilisation of certain proteins and conferring a typical salty taste by enhancing the flavour of such food products (Armenteros et al., 2009).Vacuum packaging method is a type of passive modified atmosphere.After placing the food in a suitable packaging material in this operation, the air in the package is emptied by vacuum and a hermetic closure (air tight) is made.This method is a rarely used for preservation of meat products.Even in vacuum packaging a low percentage of O 2 remain in the package and this can be used by aerobic microorganisms; with CO 2 production.In these types of products, the bacterial growth and oxidation of the product is prevented as air is not in the package (Keleş, 1998;Gülyavuz and Ünlusayin, 1999;Kilinç and Çakli, 2001).Thus, numerous studies have been conducted on the preservation of vacuum on fish and fish products of salmon (Leroi et al., 2000;González-Rodríguez et al., 2002;Martinez et al., 2010), ascidia (Stamatis et al., 2008), sardine (Senesi et al., 1980;Gómez-Estaca et al., 2010) and trout (Schulze 1985;Lyhs et al., 1998;Frangos et al., 2010;Oğuzhan and Angiş, 2012).
The aim of this study was to determine the effects of salting and packaging (vacuum and MAP) on the shelf-life of refrigerated (4°C) liquid smoking rainbow trout fillets by evaluating microbiological and chemical parameters.

Preparing samples
Rainbow trout (Oncorhynchus mykiss) (250±25 g) were obtained from Rainbow Trout Breeding and Research Center, Fisheries Department, Agricultural College, Ataturk University.Fish were carried to laboratory and washed with tap water.A total of 72 fish samples were eviscerated, stored until rigor had resolved and then filleted, 144 fillets in total (Robb et al., 2002).

Brine salting process
Fillets were washed to remove blood and mucous remains and were divided into two groups.The first and second groups were submitted to 35 and 70% brine salting (NaCl) at 4°C for 4 h, respectively.Liquid smoke flavouring (GMT Food Ingredients CO., Istanbul-Turkey) (100 ml in 1 L of brine solutions) was added into two groups and fillets were dried for 30 min.After drying, samples were smoked at 80-90°C.Fillets were packed by applying vacuum and modified atmosphere (50% CO2+50% N2).

Microbiological analysis
A sample (25 g) was taken from each group, transferred aseptically into a stomacher bag containing 225 ml of 0.1% peptone water and was homogenized for 60 s in Stomacher (Lab Stomacher Blender 400-BA 7021 Sewardmedical, England) at room temperature.For microbial analyses, 0.1 ml samples of serial dilutions (1:10, diluent, 0.1% peptone water) were inoculated onto proper agar plates.Total mesophilic aerobic bacteria (TMAB) and total psychrotrophic aerobic bacteria (TPAB) were determined on Plate Count Agar (PCA, Merck 1.05463.0500),which were incubated at 30°C for two days and at 10°C for 7 days, respectively.Pseudomonads were determined using cetrimide fusidin cephaloridine agar (CFC, Pseudomonas Agar Base-Oxoid CM0559 + CFC Selective Agar Supplement-Oxoid SR0103) after incubation at 25°C for 2 days.Lactic acid bacteria (LAB) were determined by using Man Rogosa Sharpe agar (MRS, de Man, Rogosa Sharpe Agar Oxoid CM0361), which was incubated at 30°C for 2 days.Enterobacteriaceae count were performed (Violet Red Bile Dextrose Agar Merck 1.10275.0500)which was incubated at 30°C for 2 days.Yeast and mould were determined on Rose Bengal Chloramphenicol (RBC) Agar (Merck 1.00467.0500),which was incubated at 25°C for 5 days.

Chemical analysis
Total volatile base nitrogen (TVB-N) was determined according to Malle and Tao (1987).TVB-N contents were expressed as mg 100/g fish muscle.Thiobarbituric acid reactive substances (TBARS) was determined according to the method of Lemon (1975) and Kiliç and Richards (2003).TBARS content was expressed as µmol malondialdehyde (MDA)/kg fish muscle.pH was determined according to the method of Gökalp et al. (1999).

Statistical analysis
Experiments were replicated twice on two separate occasions with different fish samples.Analyses were run in duplicate for each replicate.All obtained data from this study were subjected to analysis of variance (ANOVA), and followed by Duncan's multiple range test to determine significant differences among means at α = 0.05 level by using SPSS (1999).

Microbiological changes
Changes in TMAB of refrigerated rainbow trout fillets during storage under vacuum and modified atmosphere packaging are shown in Figure 1a.The initial count (day 0) of TMAB was 2.0 log cfu/g.V1, M1 and V2 exceeded the limit (7 log cfu/g) for fresh marine species (ICMSF, 1992) on days 90, 105 and 105 of storage, respectively.This limit was not exceeded throughout storage in M2.At 120 days, V1, M1, V2 and M2 showed 9.68, 8.29, 8.05 and 5.45 log cfu/g respectively.
The initial (day 0) psychrotrophic bacteria (Figure 1b) of rainbow trout fillets was 2.0 log cfu/g.V1, M1 and V2 exceeded the value of 7 log cfu/g for psychrotrophic bacteria, which was considered as the upper acceptability limit for fresh marine species (ICMSF, 1992) on days 90, 105 and 105 of storage, respectively.This limit was not exceeded throughout storage in M2.The counts of V1, M1, V2 and M2 were 9.65, 8.22, 8.24 and 5.83 log cfu/g at the end of storage, respectively.

TVB-nitrogen
The amount of TVB-N is an important criteriation in determining freshness of fish and their products because generally TVB-N values increase according to progression of spoilage process (Köse and Koral, 2005).TVB-N consist of TMA and ammonia with the effect of the bacteria and endogenous enzymes in fish.TVB-N value increase depend on the storage period in preservation of fish and fish products (Lannelongue, 1980;Öksüztepe et al., 2010).TVB-N values (Figure 2a) at 0 day were 18.12, 17.54, 17.99 and 16.81 mg N/100 g for V1, V2, M1 and M2, respectively.V1, M1 and M2 exceeded the limit (25 mg/100 g) for rainbow trout (Robb et al., 2002) on days 90 of storage, whereas M1 exceeded the value of 25 mg/100g on days 75 of storage.

Lipid oxidation
Lipid oxidation is one of the factor causing spoilage in product.Ransidity taste and yellow colour is seen in oxidation products (Ruiz-Capillas and Moral, 2001).Lipid oxidation is a major quality problem especially in fatty marine species.It leads to the development of off-odors and offtastes in edible oils and fat containing foods, known as oxidative rancidity.The TBARS value is an index of lipid oxidation measuring malondialdehyde (MDA) content.MDA is formed through hydroperoxides, which are the initial reaction products of polyunsaturated fatty acids with oxygen (Fernandez et al., 1998;Rezai et al., 2008).Initial TBARS values (Figure 2b) were 2.16, 2.06, 1.75 and 1.59 µmol malondialdehyde (MDA)/kg for V1, V2, M1 and M2, respectively.At the end of storage, TBARS values were 10.45, 7.22, 8.36 and 6.39 µmol malondialdehyde (MDA)/kg for treatments V1, M1, V2 and M2, respectively.pH pH value of fish meat usually ranges from 5.7-6.6.Fresh fish is close to neutral pH, after death, lactic acid will be formed firstly; falling and then rising again with spoilage (Bilgin et al., 2007).The pH values of rainbow trout fillets (Figure 2c) were 6. 15, 6.11, 6.15 and 6.02 (at initial experiment) and 6.5, 6.48, 6.42 and 6.41 (at end of storage) for V1, V2, M1 and M2, respectively.

DISCUSSION
Similar findings were reported by other researches (Dodds et al., 1992;Göktepe and Moody, 1998;Hansen et al., 1998;Kolsarici and Özkaya, 1998;Leroi et al., 1998;Paludan-Müller et al., 1998;González-Rodríguez et al., 2002;Dondero et al., 2004;Çakli et al., 2006;Erkan, 2012).Bacterial growth of modified atmosphere packaged samples was lower than vacuum packaged samples, probably, due to the presence of CO 2 gas in MAP.The carbon dioxide can be considered effectively inhibitory on the total mesophilic and psychrotrophic aerobic bacteria.Similar results were observed by several researchers (Leroi et al., 1998;Dondero et al., 2004;Çakli et al., 2006;Gümüş et al., 2008;Kilinç et al., 2009;Erkan, 2012;Frangos et al., 2010;Prygotou et al., 2010;Can, 2011;Oğuzhan and Angiş, 2012).Pseudomonas spp.were recognised early as putative spoilage inducers in fish muscle and have since then been found in various fish species from fresh and marine waters as well as in other foods (Castell and Greenough, 1957;Macdonell and Colwell, 1985;Reynisson et al., 2009).This result is due to liquid smoking.Pseudomonas was seen in the duration of storage in all groups.Similar findings were observed by Jay (1986) for hot smoked roach and whitefish.
LAB are facultative anaerobic bacteria that can grow under both anaerobic and aerobic conditions (Plahar et al., 1991).The initial LAB numbers 2.0 log cfu/g while this values increased during storage time in all groups.Similar results were found for hot smoking rainbow trout (Kolsarici and Özkaya, 1998;Çakli et al., 2006).
Enterobacteriaceae, a hygiene indicator, was also part of the microflora of fresh rainbow trout.Similar findings were observed for cold smoking salmon by Hansen et al. (1998).Similar yeast and mould (day 0) were reported for herring by Plahar et al. (1991) and Kaya et al. (2006).
TBA values represent the degree of the rancidity in the products and fresh fish is much lower than the acceptable upper limits of 8 mg malonaldehyde/kg (Schormüller et al., 1969).In "perfect material", TBA value should be less than 3 mg malonaldehyde/kg, in " good material" TBA value should not be more than 5 mg malonaldehyde/kg and consumption limit for TBA value is between 7 and 8 mg malonaldehyde/kg.
TBA values showed the degree of rancidity in the products, and values greater than 3-4 mg malonaldehyde/kg indicated a loss of product quality (Frangos et al., 2010).This values increased in the duration of storage time in all groups.Similarly, TBARS values have been reported for mirror carp (Duman an Patir, 2007), mullet (İbrahim et al., 2008), atlantic bonito (Koral et al., 2010) and salmon (Martinez et al., 2010).
The pH in fresh fish flesh is almost neutral.In the postmortem period, decomposition of nitrogenous compounds leads to an increase in pH in the fish flesh.The increase in pH indicates the loss of quality (Can, 2011).Similarly, pH values have been reported for chub mackerel (Goulas and Kontominas, 2005;Chatzikyriakidou and Katsanidis, 2012), salmon (Martinez et al., 2010) and rainbow trout (Alçiçek, 2011).pH values of modified atmosphere packaged group were lower than that of vacuum packaged samples.This result is due to carbonic acid conversion of carbon dioxide.