High Pressure Research, 2014Vol. 34, No. 1, 110–121, http://dx.doi.org/10.1080/08957959.2013.836643

The effect of chitosan-based edible film and high hydrostaticpressure process on the microbiological and chemical quality ofrainbow trout (Oncorhynchus mykiss Walbaum) fillets during

cold storage (4 ± 1◦C)

Ali Günlüa∗, Sinem Sipahioglub and Hami Alpasc

aFaculty of Fisheries, Mugla Sıtkı Koçman University, Mugla, Turkey; bAntalya Aquarium, Antalya,Turkey; cFood Engineering Department, Middle East Technical University, Ankara, Turkey

(Received 17 June 2013; final version received 29 July 2013)

The objective of this study is to determine the changes in the chemical and microbiological quality of freshrainbow trout (Oncorhynchus mykiss Walbaum) fillets during storage at 4 ± 1◦C as a result of chitosan-based edible film coating, vacuum packaging and high pressure application processes. Chemical (pH,total volatile basic nitrogen and thiobarbituric acid index) and microbiological (total mesophilic and totalpsychrophilic microorganism) shelf life analyses were carried out in 4-day intervals for samples thatwere vacuum packaged (C), subjected to high pressure after vacuum packaging (high hydrostatic pressure(HHP)), vacuum packaged after being wrapped by chitosan-based film (CFW) and subjected to highpressure after vacuum packaging and being wrapped by chitosan-based film (HHP + CFW). According tothe chemical and microbiological shelf life analysis results of rainbow trout fillets, shelf life increases of4 days in HHP group samples, 8 days in CFW group samples and 24 days in HHP + CFW group sampleswere provided in comparison with the control group. In conclusion, it was determined that high pressureand wrapping with chitosan-based film had protective effect both chemically and microbiologically andthat the most effective protection was obtained when both methods were used together.

Keywords: Oncorhynchus mykiss; chitosan; edible film; high hydrostatic pressure; shelf life

Introduction

Edible films are thin layers made of edible material placed on or between food components toextend shelf life.[1] Thin layers can also be applied by wrapping it over the food material.[2] Ediblefilm and coatings have a range of advantages, such as edibility, biodegradability, biocompatibility,aesthetic appearance and barrier features, as well as being nontoxic and nonpolluting.[3,4] Inaddition, they are accepted as food preservatives due to the facts that they can contain antimicrobialagents, antioxidants and other food additives harmoniously when added into the film duringproduction along with their shelf life-increasing abilities.[5,6]

Chitosan is obtained from crustacean shells and is the second most frequently found nat-ural biopolymer following cellulose.[7] It has a wide application area due to its nontoxicity,biodegradability and commercial availability.[8,9] This biopolymer has a perfect ability to formfilm and coating solution when dissolved in organic acid aqueous solutions.[10] Hence, it has a

∗Corresponding author. Emails: aligunlu@mu.edu.tr, alifuatgunlu@gmail.com

© 2013 Taylor & Francis

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wide potential for use as a food packaging material.[11–13] Studies carried out on various fishspecies have put forth that chitosan-based film and coatings decrease microorganism development,enhance fish quality and increase storage period.[12,14–22]

High hydrostatic pressure (HHP) process is one of the non-thermal processing technologieswith wide consumer acceptance due to its high reliability and quality in comparison with otherthermal and non-thermal technologies along with the fact that it does not require the use ofadditive materials.[23] HHP is among the newly developed methods to increase the safety andshelf life of easily spoiled foods by preventing the development of vegetative bacteria that arefound in many foods causing spoilage and pathogen.[24,25] There are many studies stating thatmicrobiological and chemical quality has been increased as a result of HHP application on octopusarm muscle,[26] cold-smoked sardines,[15] rainbow trout and mahi mahi,[27] rainbow trout,bream and mullet,[28–30] cold-smoked salmon and Atlantic horse mackerel,[31,32] ringa andred mullet [33] and shrimps,[34]

Rainbow trout is a species of aquaculture fish that is bred intensively in the river and damlakes of Turkey and sold either as fresh frozen or as fillet in the local market. In addition, it isalso widely exported to EU countries either as fresh frozen whole – fillet or hot-smoked vacuumpackaged.[35] Since rainbow trout is highly consumed in both the local and the internationalmarkets, the increase in its normally rather short shelf life in refrigerated conditions arises as arather significant problem. Many studies have been conducted to determine the shelf life of theaquaculture rainbow trout during refrigeration storage.[21,36–40] Mexis et al. [36] have statedthat the shelf life of rainbow trout fillets stored at 4◦C is 4 days according to the sensory andmicrobiological analysis results. Whereas in another study, the shelf life of rainbow trout storedin refrigerator conditions after being vacuum packed has been reported as 10 days.[40] Similarly,it has been determined by other researchers that the shelf life of rainbow trout stored at 4◦C rangesbetween 9 and 15 days.[21,37–39]

The increase in consumer demands regarding high-quality, reliable fish meat with no additivesand a high shelf life has resulted in the increase in studies on the processing and packagingprocesses of fish meat. Until now, studies carried out to increase the shelf life of fish meat havebeen limited to wrapping with chitosan-based edible film, followed by vacuum packaging andHHP applications. The objective of this study is to determine the combined effect of chitosan-based film wrapping, vacuum packaging and HHP process on the shelf life of rainbow trout filletduring the storage at 4◦C.

Material and method

Material

In this study, 80 rainbow trout (Oncorhynchus mykiss) obtained from aquaculture producers(Baysallar-Isparta-Turkey) with an average weight of 250–300 g were used. Eighty rainbow troutwere placed in tanks filled with ice-cold water and they were killed rapidly. The fish were put into styrofoam boxes and covered with bags filled with dry ice and then they were taken into thefood laboratory within 1 h. The internal organs of the samples were cleaned in the laboratory andwashed with water after which their skinless fillets were taken and the fillets were cut from theirsides into orderly rectangles of equal weight (about 10 g).

Preparation of chitosan-based edible films

Chitosan-based edible films were prepared as described by Günlü and Koyun.[22] In order toobtain chitosan-based edible biofilms, commercial chitosan obtained from shrimp shells (C3646)

112 A. Günlü et al.

was used. About 1.5 g of chitosan was weighed and 100 mL of 1% acetic acid was added in orderto obtain biofilm. The solution was mixed at 40◦C for 30 min in a magnetic mixer (Wisestir, MSH20A, Korea) to ensure that the chitosan dissolves completely in acetic acid, after which 1.5 gglycerol (Sigma G8773) was added as a plasticizer and the mixing was continued in the magneticmixer at the same temperature for the same duration. The solution was transferred to the buchnerfunnel in order to remove the air bubbles that form inside the obtained edible film solution andthe air bubbles were removed via a vacuum pump. One hundred millilitres of the film solutionwas taken and placed on styrofoam plates (20 × 10 cm) which were placed in a dry air sterilizer(Labart, DHG 9140A, Korea) and held at 40◦C for 8 h, cooled at room temperature and the coatingfilms were taken out of the plates using pincers. The films that are very flexible and brittle werekept in a dessicator until the wrapping process of the fillet pieces was completed.

Wrapping of the fish with film and trial plan

Fillets that were previously cut into small pieces (10 g) were separated into four groups. The filletpieces in the first group were only packaged using a polyethylene film (Ayhan Plastic Limited,Isparta, Turkey) in vacuum (Abant Vacuum Systems, Turkey) (Control group-C). The fillet piecesin the second group were packaged in vacuum and HHP was applied. Whereas the fillet pieces inthe third group were coated with chitosan film separately, the tips of the biofilms were squeezedafter which they were packed under vacuum by placing in vacuum bags (CFW). The fillet piecesin the last group were wrapped by chitosan-based film separately, wrapped in vacuum and thenwere subject to HHP process (HHP + CFW) (Table 1).

HHP application

HHP treatments were performed in a designed and constructed laboratory-scale unit (capacity:30 cm3, maximum pressure: 500 MPa). The equipment consisted of a pressure chamber of cylin-drical design, two end closures, a means for restraining the end closures, a pressure pump and ahydraulic unit to generate high pressure for system compression and also a temperature controldevice. The pressure vessel was made of hot galvanized carbon steel and the piston was hardchrome-plated and polished to mirror finish (steel-type heat-treated special K) which was pro-cessed into the required sizes at the Electrical and Electronic Engineering Department of MiddleEast Technical University (Ankara, Turkey). The liquid was heated up to the desired temperatureprior to pressure application using an electrical heating system encompassing the cell.[41]

The vacuum-packed fillet pieces which will be subjected to the HHP process were wrappedwith a stretch film tightly and brought to the Middle East Technical University Food EngineeringDepartment within 10 h after being placed in styrofoam boxes with dry ice and were then subjectedto high pressure process in the HHP Unit under 220 MPa at 15◦C for 5 min. Motorin oil was usedas a fluid in the pressure unit for pressure transmission.

Table 1. Trial plan.

Trial groups CODE

Control (vacuum packed) CHigh pressure + vacuum packed HHPVacuum packed + chitosan-based film wrapping CFWHigh pressure + chitosan-based film wrapping + vacuum packed HHP + CFW

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The storage of samples

All the fillet pieces in the trial groups were placed in +4 ± 1◦C coolers and subjected to analysesat 4-day intervals in order to carry out the shelf life and other quality analyses.

Quality control analyses

pH analysis

Ten times (45 mL) pure water was added to the muscle samples that were cut into small pieces(5 g) and pH values were measured using a pH meter (WTW 330i, Almanya) following a homog-enization of 1 min in a blender (Waring Blender, USA). The pH meter was calibrated using 4.00and 7.00 buffer solutions prior to the measurement.[42]

Microbiological analyses

Twenty-five grams of samples that were taken using sterile pincers, scalpel and scissors underaseptic conditions were homogenized for 60 s using 225 mL sterilized buffer pepton water andstomacher (BackMikser 400, France), and dilution fluids up to 10−6 were prepared. Microbio-logical inoculations were carried out according to the pour-plate method. The total mesophilicmicroorganism (TMM) count was determined using plate count agar (PCA) at the end of the 72 hincubation at 30 ± 1◦C, total phsychrophilic microorganism (TPM) count was obtained usingPCA as a result of 10-day incubation at 4 ± 1◦C. The results were given as the logarithm of thecolony number formed in each gram sample (log cfu/g).[43,44]

Determination of thiobarbituric acid index (TBA-i) value

Thiobarbituric acid index (TBA-i) value was determined as described by Erkan et al.[45] Twograms of the sample was weighed using the precision scale after which 0.1% 100 μL butylatedhydroxytoluene (ethanol 1 g/L) (Fluka 34750) and 5% 25 ml trichloroacetic acid (Riedel-de Haen27242) were added and homogenized at medium rotation for 2 min using Ultra-Turrax (Heidolph,DIAX 900, Germany). The mixture was filtered with a Whatman No 1 filter paper and 5 mLof the filtrate was taken and 5 mL of the newly prepared TBA reactive (0.02 M of the solutionof 2-thiobarbituric acid in 90% acetic acid) (Merck 108180) was added and the cap was tightlyclosed. The tubes were kept in water bath (Memmert, WB 22, GERMANY) at 95◦C to ensure thatthe reaction starts. The same process was applied on MDA standard (1,1,3,3-tetraethoxypropan(Merck 820756) and blank. The standards and samples were read with respect to the blank at532 nm after the tubes were cooled down. The absorbances that were read in the spectrophotometerwere calculated using the regression curve of the standards and TBA-i (μg malondialdehyde[MDA]/mL) concentration was calculated from dilution factor/samples weight. The TBA-i valuewas expressed as milligram MDA equivalents per kilogram of fish muscle.

Total volatile basic nitrogen (TVB-N) value

The total volatile basic nitrogen (TVB-N) amounts were determined according to the methodstated by Antonacopoulos and Vyncke.[46] Ten grams of fish muscle was homogenized with 6%90 mL of perchloric acid (Merck 100518) for 1 min in a blender (Waring, USA). The homogenatewas filtered using a filter paper (Whatman no. 1). Fifty millilitre of the filtrate was taken priorto the vapour distillation process, 7 drops of phenolphthalein was dripped and using sufficient

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20% NaOH (Sigma-Aldrich 06203) the formation of purple violet was noted. In addition, a fewdrops of the foam inhibitor were added to the filtrate during distillation in order to prevent foamformation. One hundred millilitres of 3% boric acid solution was placed inside the distillatecollection erlenmeyer flask and the distillation process was completed using a fully automatedvapour distillation device for 10 min to collect 100 mL of the distillate. The distillate collectedinside the boric acid was titrated with 0.01 N HCl (Sigma-Aldrich 07102) using an automatictitrator with 5 taken as the pH equivalence point after which the TVB-N amount was calculatedusing the formula below and the total consumed HCl

TVB − N(mg/100 g) = (HCl consumption × 0.14 × 2 × 100)

Sample weight.

Statistical analyses

All experiments were performed in triplicate. The averages of the analyses’ results were givenwith standard deviations. The data obtained as a result of the study were subjected to varianceanalysis (F test) using SPSS 16.0 Windows software and the averages of the significant variancesources were selected and compared via Duncan’s Multiple Comparison Test with a significancelevel of P = 0.05.

Results and discussion

pH analyses results

The activity of each microorganism and enzyme increases at a certain pH value. The changesthat occur in the pH value of the C, HHP, CFW and HHP + CFW group rainbow trout filletsstored in refrigerator conditions (4 ± 1◦C) are given in Figure 1. The initial pH value at thebeginning of storage was specified as 6.56 ± 0.02 for the C group, as 6.60 ± 0.08 for the HHPgroup, as 6.57 ± 0.03 for the CFW group and as 6.44 ± 0.18 for the HHP + CFW group. Thelowest pH value among the studied samples was determined in the HHP + CFW group on the 44thday, whereas the highest pH value was determined in the C group on the 16th day. Significantincrease (P ≤ 0.05) in the pH value of the control group due to storage has been determined,whereas insignificant changes (P ≥ 0, 05) have been determined for other groups (Figure 1).The acceptable upper limit value of the pH value of the fish has been specified as 6.8–7.0.[47]According to our study results, this limit value has not been exceeded in any trial group duringstorage at 4◦C.

Microbiological analysis results

The TMM count for the determination of the microbiological quality of the rainbow trout filletsstored in refrigerator conditions is given in Figure 2, whereas the TPM count is given in Figure 3.The TPM counts at the beginning of storage were determined to be in the interval of 1.16–2.99 log cfu/g, whereas the TMM counts were found to be in the interval of 1.84–3.57 log cfu/gaccording to the results of the study. Significant (P ≤ 0.05) increases have been determined in allgroups during storage (Figure 2). Psychrophilic bacteria make up the most important microorgan-ism group responsible from the aerobic spoilage of fresh fish stored at low temperatures.[48] Theacceptable limit values for the psychrophilic and mesophilic aerobic microorganisms’ count infresh fish have been put forth by different researchers as 6 log cfu/g [43,49] and 7 log cfu/g,[48]respectively. The acceptable limit values (6 log cfu/g) for TMM and TPM count exceeded on the

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Figure 1. Changes in pH values in rainbow trout fillets during storage at 4◦C, when vacuum packaged (C), highhydrostatic pressure applied after vacuum packaged (HHP), vacuum packaged after wrapped with the chitosan-basededible film (CFW) and high pressure applied after vacuum packaging and wrapping with the chitosan-based edible film(HHP + CFW). Different lowercase letters (a, b and c) represent significant differences between sampling dates for agiven group. Different capital letters (A, B and C) represent significant differences between groups on different samplingdates.

12th, 16th, 24th and 36th days for C, HHP, CFW and HHP + CFW, respectively. It has been statedby Jeon et al. [14] that as a result of the storage of chitosan-wrapped ringa and morina fish in thecold for a period of 12 days, a decrease of 3 and 2 log, respectively, occurred in the microbialload. Similar results have been obtained in many studies carried out on the effectivity of chitosan-based coating films regarding the prevention and decrease of the microbial spoilage.[16–22] Inaddition, Gómez-Estaca et al. [15] have reported that gelatine–chitosan films are very effectivein the prevention of microbial development in cold-smoked sardine fish. Similarly, the effective-ness of HHP process on the prevention and decrease of microbial development in stored fish hasbeen examined for many fish species. Significant decrease in microbial load as a result of HHPapplication has been put forth by Hurtado et al. [26] for octopus and octopus arm muscles, byGómez-Estaca et al. [15] for cold-smoked sardines, and by Büyükcan et al. [41] for shrimps andking scallops. While Gómez-Estaca et al. [15] have determined that the microbial load decreasedsignificantly in comparison with the control samples during the first period of cold storage fol-lowing HHP process on cold-smoked Coryphaena hippurus fillets no significant changes weredetermined in the microbial load towards the end of the shelf life.

TBA-i value results

TBA-i is an important indicator of lipid oxidation in meat. TBA-i value is determined by mea-suring the MDA which is a spoilage product of the lipid hyperoxides that form as a result ofthe oxidative processes of unsaturated fatty acids.[50] Lipid oxidation is a significant prob-lem relative to off-flavour and off-odour development in fish muscle which typically containsa high percentage of polyunsaturated fatty acids.[51] According to Connell,[52] TBA values of1–2 mg MDA/kg of fish flesh are usually regarded as the limit beyond which fish will normallydevelop an objectionable odour.

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Figure 2. Changes in TMM count in rainbow trout fillets during storage at 4◦C, when vacuum packaged (C), highhydrostatic pressure applied after vacuum packaging (HHP), vacuum packaged after wrapping with the chitosan-basededible film (CFW) and high pressure applied after vacuum packaging and wrapping with the chitosan-based edible film(HHP + CFW). Different lowercase letters (a, b and c) represent significant differences between sampling dates for a givengroup. Different capital letters (A, B and C) represent significant differences between groups on different sampling dates.

Figure 3. Changes in TPM count in rainbow trout fillets during storage at 4◦C, when vacuum packaged (C), highhydrostatic pressure applied after vacuum packaged (HHP), vacuum packaged after wrapping with the chitosan-basededible film (CFW) and high pressure applied after vacuum packaging and wrapping with the chitosan-based edible film(HHP + CFW). Different lowercase letters (a, b and c) represent significant differences between sampling dates for a givengroup. Different capital letters (A, B and C) represent significant differences between groups on different sampling dates.

The lowest and highest TBA-i values during storage were determined on the 4th and 16th daysof storage 0.32 ± 0.01 − 2.80 ± 0.59 for fresh samples (C). Whereas no significant change in theTBA-i value was observed on the 16th day of storage among HHP, CFW and HHP + CFW groups,

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Figure 4. Changes in TBA-i values in rainbow trout fillets during storage at 4◦C, when vacuum packaged (C), highhydrostatic pressure applied after vacuum packaging (HHP), vacuum packaged after wrapping with the chitosan-basededible film (CFW) and high pressure applied after vacuum packaging and wrapping with the chitosan-based edible film(HHP + CFW). Different lowercase letters (a, b and c) represent significant differences between sampling dates for agiven group. Different capital letters (A, B and C) represent significant differences between groups on different samplingdates.

a significant increase was observed in the C group in comparison with these groups (P ≤ 0.05).The highest TBA-i value for the HHP and CFW groups was determined on the 24th day of storage.Significant decreases (P ≤ 0.05) were observed during storage in the HHP + CFW group, whereassignificant (P ≤ 0.05) increases were observed in other groups (Figure 4). Similar to our findings,it has been put forth by many researchers that lipit oxidation decreases significantly as a result ofthe coating of fish meat with chitosan.[12,14–16,18,19] The limit TBA-i (1–2 mg MDA/kg) valueput forth by Connell [52] was exceeded during cold storage by C, HHP and CFW on the 16th, 24thand 24th days, respectively. Whereas the TBA-i value of the HHP + CFW group was determinedto be below the limit value even on the 44th day of storage. Gómez-Esteca et al. [15] have appliedhigh pressure (300 MPa/20◦C/15 min) and marjoram extract or gelatine-based functional ediblefilms enriched by rosemary or chitosan either one by one or in combination in order to increasethe shelf life of cold-smoked sardines (Sardina pilchardus) and it has been stated that the bestresult for the prevention of lipid oxidation was obtained in high pressure – chitosan-based ediblefilm combination. This result is in accordance with our results. The highest TBA-i value at thebeginning of storage was determined in the HHP + CFW group followed by the HHP group. Inaccordance with our results, it has also been stated in other studies that lipid oxidation increasedsignificantly due to high pressure application on fish meat.[25,53,54]

TVB-N analyses results

One of the most widely used variables in determining the freshness of fish and fish productsis the TVB-N value. The amount of volatile basic nitrogen substances increases along with theadvancement of spoilage in fish. This increase is in relation with spoilage bacteria and endogenousenzyme activity.[55,56] The acceptable TVB-N limit values differ from fish to fish. Different limitvalues have been determined in many studies and it has been stated that these values range between

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Figure 5. Changes in TVB-N values in rainbow trout fillets during storage at 4◦C, when vacuum packaged (C), highhydrostatic pressure applied after vacuum packaging (HHP), vacuum packaged after wrapping with the chitosan-basededible film (CFW) and high pressure applied after vacuum packaging and wrapping with the chitosan-based edible film(HHP + CFW). Different lowercase letters (a, b and c) represent significant differences between sampling dates for agiven group. Different capital letters (A, B and C) represent significant differences between groups on different samplingdates.

20 and 35 mg/100 g.[52,57–59] According to Sikorski et al.,[60] it has been stated that the 30 mgTVB-N/100 g level in fish meat is an indication of spoilage. Ludorff and Meyer [47] have putforth that the acceptable upper limit for sea food is 30–35 mg TVB-N/100 g. Significant increases(P ≤ 0.05) have been observed in the TVB-N values of all trial groups during cold storage. Thehighest value was obtained in the control group on the 16th day as (45.77 ± 0.50), while thelowest value was obtained again in the control group on the 4th day (18.67 ± 0.48) samples(Figure 5). Similar to the initial TVB-N value determined for fresh samples at the beginning ofstorage, the TVB-N value for fresh rainbow trout has been stated to be between 12.42 mg/100 gand 22.51 mg N/100 g by other researchers.[40,61–63] The acceptable limit value for TVB-N specified by Sikorski et al. [60] (30 mg TVB-N/100 g) has been exceeded in the C, HHP,CFW and HHP + CFW groups on the 12th, 20th, 24th and 44th days, respectively, during coldstorage. The decrease in the TVB-N value observed in fish meat in the control group due to coldstorage was also put forth by other researchers for both high pressure applied [15,29,33,41,64]and chitosan-based edible film coating or wrapped [14,16,19–22,65,66] fish meat.

Results

In this study which aimed to determine the effects of chitosan-based edible film wrapping andHHP processes separately and jointly on the chemical and microbiological quality of rainbowtrout fillets, it has been determined that high pressure and chitosan-based film wrapping havepositive effects on quality while the combined application of the two methods provides betterprotection. It has been determined as a result of the microbiological shelf life analyses carried outthat the acceptable limit values for TMM and TPM counts of the fillets exceeded on the 12th, 16th,24th and 36th days of storage in C, HHP, CFW and HHP + CFW group samples, respectively,

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as a result of chemical shelf-life analysis that the TVB-N value exceeded on the 12th, 20th, 24thand 44th days, respectively, and that the TBA-i value exceeded in the first three groups on the16th, 24th and 24th days whereas it did not exceed in the last group even on the 44th day. In thelight of microbiological analyses, a shelf-life increase of 4 days for the fillets as a result of highpressure application process, 8 days as a result chitosan-based wrapping process and 24 days asa result of the combined usage of both processes have been found.

In conclusion, it is thought that the microbial and chemical qualities of fish and fish productscan be preserved for a longer period of time as a result of chitosan-based edible film wrappingand high pressure application processes, thereby presenting the consumers with reliable productswith no additives.

Acknowledgements

This study is a summary of the Master’s Thesis of Sinem Sipahioglu. We would like to thank Süleyman Demirel UniversityScientific Studies Management Unit Council for their financial support with 2107-YL-10 numbered project.

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