International Journal of Food Science and Technology 2010, 45, 1717–1723 Original article The capability of rosemary extract in preventing oxidation of fish lipid Yesim Ozogul,1* Deniz Ayas,2 Hatice Yazgan,1 Fatih Ozogul,1 Esmeray K Boga1 & Gulsun Ozyurt1 Department of Seafood Processing Technology, Faculty of Fisheries, University of Cukurova, Adana, Turkey Department of Seafood Processing Technology, Faculty of Fisheries, University of Mersin, Mersin, Turkey (Received 11 January 2010; Accepted in revised form 26 May 2010) Summary The effects of rosemary extract at different levels (%1, R1, and %2, R2) on the quality of vacuum-packed sardine in terms of sensory, biochemical (thiobarbituric acid, total volatile basic nitrogen, peroxide value and free fatty acids) and microbiological analyses (total viable counts) were investigated Fish were filleted and divided into three groups First group was used as the control (C) without rosemary extract, second group was treated with 1% rosemary extracts (10 g L)1) for (R1), and the third was treated with 2% rosemary extracts (20 g L)1) for (R2) Thirty fillets per litre were used After that, all groups were vacuum-packed in polyethylene bags The samples were stored in the refrigerator condition (4 ± °C) over the storage period of 20 days The results showed that the use of rosemary extract improved the sensory quality of both raw and cooked sardine, most preferably sardine treated with 1% of rosemary Biochemical analysis showed that the use of 2% of rosemary extract were found to be most effective (P < 0.05) in controlling the rate of lipid oxidation Keywords Lipid oxidation, quality, rosemary extract, sardine, vacuum package Introduction Fish is one of the most highly perishable food products, and the shelf life of such products is limited in the presence of atmospheric oxygen and the growth of aerobic spoilage microorganisms Fish oil contains longchain polyunsaturated fatty acids (PUFA) such as EPA (eicosapentaenoic acid, C20:5n3) and DHA (docosahexaenoic acid, C22:6n3) that are considered to have a number of health benefits However, the desirable PUFA content in fish oil is highly vulnerable to oxidative destruction Lipid oxidation is a series of chain reaction with molecular oxygen reacting with unsaturated lipids to form lipid peroxides resulting in organoleptic changes of flavour, texture and aroma of food (Sarkardei & Howell, 2008) Synthetic antioxidants such as ethylenediaminetetraacetic acid, butylated hydroxyanisole and butylated hydroxytoluene can be added to retard fish oil oxidation, but antioxidants from natural sources may be used to replace synthetic antioxidants Recently, there is an increasing demand for natural antioxidants because of the concern about safety of synthetic antioxidants This would not only *Correspondent: Fax: (90) 322 3386439; e-mail:yozogul@cu.edu.tr prevent omega-3 fatty acid oxidation, but also enhance the health benefits of the foods by having the additional health-promoting bioactivity from the herbs or spices It was also reported that rosemary extracts contain a large amount of phenolic compounds such as carnosic acid, carnosol and rosmarinic acid, which have antioxidant potential (Frankel, 1999) Moreover, Tironi et al (2009) found that application of rosemary extract at doses of 200 and 500 ppm prevents lipid oxidation of chilled sea salmon Corbo et al (2008, 2009b) have tested thymol, lemon extract and grape fruit seed extract, at 20, 40 and 80 ppm, against the main spoilage microorganisms inoculated in fish burgers stored at °C Results showed that all compounds have effect in slowing down the growth of microorganisms, suggesting that they can be advantageously used to prolong the shelf life of fresh fish burger The combined effect of modified atmosphere packaging (MAP: 40% CO2 ⁄ 30% O2 ⁄ 30% N2) and oregano essential oil on the shelf life of lightly salted cultured sea bream fillets stored under refrigeration was studied by Goulas & Kontominas (2007) They found that oregano essential oil in combination with MAP and light salting was the most effective treatment for the preservation of sea bream fillets followed by MAP doi:10.1111/j.1365-2621.2010.02326.x Ó 2010 The Authors Journal compilation Ó 2010 Institute of Food Science and Technology 1717 1718 Rosemary extract in preventing oxidation Y Ozogul et al Mahmoud et al (2006) established a new technology, using pretreatment with electrolysed NaCl solutions and essential oil compounds, to extend the shelf life of carp fillets Samples of skinless carp fillets were treated with 100-fold (by weight) of electrolysed NaCl solutions [cathodic solution, EW()), and ⁄ or anodic solution, EW(+)] and 1% oil (0.5% carvacrol + 0.5% thymol) [1%(C + T)] Results indicated that treatment with EW()) ⁄ EW(+) ⁄ 1%(C + T) extended the shelf life of carp fillets to 16 and 1.3 days compared with and 0.3 days for the control samples during storage at and 25 °C, respectively However, it was also reported that fish preservation, using electrolysed NaCl solutions and 1% (carvacrol + thymol), did not affect the quality (nutritional components) of carp fillets and could be a good alternative to synthetic preservatives used in the food industry (Mahmoud et al., 2007) Sardine is commercially important fish species in Turkey as they are caught in large amounts (17.531 tons in 2008) (Anon., 2008) Sardine are generally consumed as fresh, canned or used as fish meal and oil in Turkey There are many researches on the quality of sardine stored in ice (Campos et al., 2005) and under MAP and vacuum packed (VP) conditions (Oăzogul et al., 2004; Mendes et al., 2008) The use of rosemary as antioxidant in different fish and fish products has also been reported (Akhtar et al., 1998; Gimenez et al., 2004, 2005; Oăzogul et al., 2009) However, no information is available on the effects of different level of rosemary extract on the quality of vacuum-packed sardine stored at °C The principal objectives of this investigation were (i) to determine the shelf life of the sardine treated with different levels of rosemary extract; (ii) to evaluate some of the existing objective tests as indices of quality and degree of spoilage of vacuum-packed sardine; and (iii) to determine antioxidant effects of rosemary extract on vacuum-packed sardine (Sardinella pilchardus) fillets during refrigerated storage Materials and methods Rosemary extract and preparation of fish Rosemary (Rosmarinus officinalis) used in this project was a powder and presented by Frey-Lau Company (Henstedt-Ulzburg, Germany) Rosemary extract was applied to the fish as described by Akhtar et al (1998) Fish (26.97 ± 1.49 g and 14.13 ± 0.31 cm) were caught by seine net in Mersin Bay, Turkey The duration of time between harvesting and arrival of the fish at the laboratory was 0.05) were found between samples treated with rosemary extract The use of rosemary extract improved the sensory quality of sardine Similar results were obtained from the other studies (Vareltzis et al., 1997; Akhtar et al., 1998; Gimenez et al., 2004, 2005) It was reported that the progress of decomposition showed off-odour after days for sardine in VP (Oăzogul et al., 2004) and days for herring in VP (Oăzogul et al., 2000) In this research, shelf life of sardine (the control) without rosemary extract was 13 days The reason for this longer shelf life is that fish were iced immediately after harvesting and also the short time between catch and storage ( 0.05) until day After that, significant differences (P < 0.05) were found between the control and treatment groups Significant differences were also observed (P < 0.05) between R1 and R2 on day 17 and 20 Off-flavour and off-odour of the control group, R1 and R2 were detected on 13, 17 and 20 day of storage, respectively, as found for raw sardine by QIM Because the flavour and taste of rosemary extract was much stronger in R2 than in R1, R1 group was mostly preferred by the panellists Chemical assessment The proximate composition of the sardine was the following: 20.60 ± 0.55% protein, 9.15 ± 0.84% lipid, International Journal of Food Science and Technology 2010 1719 1720 Rosemary extract in preventing oxidation Y Ozogul et al Table Sensory analyses of cooked sardine in VP Storage days 10 13 17 20 Colour X Ỉ Sx 9.00 9.00 9.00 8.57 8.43 8.71 7.57 7.57 7.57 7.00 7.57 7.57 5.43 6.57 6.71 1.86 3.29 7.00 1.57 2.71 4.00 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± a 0.00 0.00a 0.00a 0.54a 0.54a 0.49a 0.54a 0.54a 0.54a 0.00a 0.54b 0.54b 1.13a 0.54b 0.49b 0.90a 0.49b 0.00c 0.54a 0.49b 0.00c Odour X Ỉ Sx 9.00 9.00 9.00 9.00 8.86 9.00 7.57 8.00 8.29 6.57 7.57 8.00 4.14 6.86 7.00 2.29 3.57 6.43 1.29 2.29 3.71 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± a 0.00 0.00a 0.00a 0.00a 0.38a 0.00a 0.54a 0.00ab 0.49b 0.54a 0.54b 0.00b 0.90a 0.38b 0.00b 0.49a 0.54b 0.54c 0.49a 0.49b 0.49c Taste X Ỉ Sx 9.00 9.00 9.00 8.86 8.86 9.00 7.57 8.00 8.57 6.57 7.86 8.00 4.00 6.57 6.71 2.29 3.57 6.43 1.00 2.71 3.71 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Firmness X Æ Sx a 0.00 0.00a 0.00a 0.38a 0.38a 0.00a 0.54a 0.00a 0.54b 0.54a 0.38b 0.00b 0.82a 0.54b 0.49b 0.49a 0.54b 0.54c 0.00a 0.49b 0.49c 9.00 9.00 9.00 8.86 9.00 9.00 7.57 7.57 8.29 6.57 7.29 7.71 4.14 6.86 6.71 1.57 3.57 6.43 1.00 2.14 3.43 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± General acceptance X Ỉ Sx a 0.00 0.00a 0.00a 0.38a 0.00a 0.00a 0.54a 0.54a 0.49b 0.54a 0.49b 0.49b 0.90a 0.38b 0.49b 0.54a 0.54b 0.54c 0.00a 0.90b 0.98c 9.00 9.00 9.00 8.86 9.00 9.00 7.57 8.00 8.00 6.57 7.86 8.00 3.71 6.86 7.00 1.57 3.57 6.43 1.00 2.00 3.71 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Groups a 0.00 0.00a 0.00a 0.38a 0.00a 0.00a 0.54a 0.00b 0.00b 0.54a 0.38b 0.00b 0.49a 0.38b 0.00b 0.54a 0.54b 0.54c 0.00a 0.00b 0.49c C R1 R2 C R1 R2 C R1 R2 C R1 R2 C R1 R2 C R1 R2 C R1 R2 Different letters in the same column for each storage days indicate significant differences (P < 0.05) X Ỉ Sx , average ± standard deviation; C, control; R1, 1% rosemary; R2, 2% rosemary 68.66 ± 0.35% moisture and 1.33 ± 0.23% ash Variations in chemical composition of sardine, mainly in lipid and moisture, were reported (Oăzogul et al., 2004; Erkan & Ozden, 2008; Mendes et al., 2008) The variation in the chemical composition of fish is related to nutrition, living area, fish size, catching season, seasonal and sexual variations as well as other environmental conditions Because sardine contains high level of lipid, care must be taken to preserve the quality as fresh as possible after harvesting TVB-N is a product of bacterial spoilage and endogenous enzymes action, and its content is often used as an index to assess the keeping quality and shelf life of products (EEC, 1995) TVB-N is a general term that includes the measurement of TMA, dimethylamine, ammonia and other volatile basic nitrogenous compounds associated with seafood spoilage (Huss, 1995) In this study, TVB-N concentrations of all groups are shown in Table TVB-N content of all groups increased with storage time The maximum permissible level of TVB-N in fish and fishery products is 35 mg 100 g)1 (EEC, 1995) At the beginning of storage, the initial TVB-N value was 20.59 mg 100 g)1 flesh and increased to 34.29 mg TVB-N 100 g)1 at day 13 for the control, 33.64 mg TVB-N 100 g)1 at day 17 for R1 and 35.82 mg TVB-N 100 g)1, in which all samples in VP were rejected by the sensory panellists The lowest TVBN value (P < 0.05) was obtained from R2 followed by R1 and the control during storage period In this study, International Journal of Food Science and Technology 2010 Table Changes in the value of total volatile basic nitrogen in sardine during storage period Control Storage days 10 13 17 20 R1 X Ỉ Sx 20.59 22.44 25.10 30.67 34.29 45.79 45.96 ± ± ± ± ± ± ± R2 X Ỉ Sx a 1.20 0.44b 0.12a 0.40c 0.69c 1.13b 1.42c 20.59 23.10 24.74 28.13 32.80 33.64 40.65 ± ± ± ± ± ± ± X Æ Sx a 1.20 0.32c 1.06a 0.41b 0.31b 0.50a 0.43b 20.59 21.43 24.79 25.78 27.15 33.25 35.82 ± ± ± ± ± ± ± 1.20a 0.06a 0.26a 0.80a 0.77a 0.37a 0.29a Different letters in the same row indicate significant differences (P < 0.05) X Ỉ Sx , average ± standard deviation; C, control; R1, 1% rosemary; R2, 2% rosemary when the TVB-N level exceeded the maximum value, samples were already refused by the panellists Therefore, TVB-N values correlated well with the results of sensory analyses, providing a good index for the assessment of sardine in VP Shelf life of oily fish species is limited because of the oxidation of lipid The primary product of lipid oxidation is fatty acid hydroperoxide, measured as PV Peroxides are unstable compounds, and they break down to aldehydes, ketones and alcohols that are volatile products causing off-flavour in products Ó 2010 The Authors Journal compilation Ó 2010 Institute of Food Science and Technology Rosemary extract in preventing oxidation Y Ozogul et al Table Changes in the value of peroxide value in sardine during storage period Control Storage days 10 13 17 20 R1 X Ỉ Sx 4.32 19.56 10.46 10.24 8.72 13.48 14.28 ± ± ± ± ± ± ± R2 X Ỉ Sx a 1.10 1.62b 0.71ab 1.30b 1.52a 0.92b 2.70b 4.32 11.24 8.06 9.04 8.93 14.74 10.73 Table Changes in the value of thiobarbituric acid during storage period ± ± ± ± ± ± ± Control Storage days X Ỉ Sx a 1.10 0.21a 1.97a 0.80ab 1.22a 1.37b 1.21a 4.32 9.12 11.31 7.12 8.59 10.77 9.51 ± ± ± ± ± ± ± a 1.10 1.27a 0.47b 0.90a 0.95a 0.35a 0.72a R1 X Ỉ Sx 10 13 17 20 0.79 1.93 1.79 1.46 1.11 0.98 0.90 ± ± ± ± ± ± ± R2 X Æ Sx a 0.09 0.40c 0.34b 0.16b 0.16b 0.03b 0.05c 0.79 0.90 0.65 0.66 1.49 0.92 0.80 ± ± ± ± ± ± ± X Ỉ Sx a 0.09 0.04b 0.05a 0.12a 0.12c 0.13b 0.03b 0.79 0.58 0.58 0.73 0.75 0.66 0.65 ± ± ± ± ± ± ± 0.09a 0.03a 0.05a 0.03a 0.07a 0.15a 0.03a Different letters in the same row indicate significant differences (P < 0.05) X Ỉ Sx , average ± standard deviation; C, control; R1, 1% rosemary; R2, 2% rosemary Different letters in the same row indicate significant differences (P < 0.05) X Ỉ Sx , average ± standard deviation; C, control; R1, 1% rosemary; R2, 2% rosemary (Hamilton et al., 1997) Peroxide and thiobarbituric acid (TBA) values are the major chemical indices to measure the degree of oxidative rancidity In this study, the PV of oil extracted from the sardine fillets treated with and without antioxidants increased up to days for the control and R1 and days for R2, after which the values fluctuated during storage period (Table 4) Although there were no significant differences (P > 0.05) among the groups, significant differences were observed (P < 0.05) on days and 20 between samples with antioxidant (R1 and R2) and the control without antioxidant The initial value of sardine was found, 4.32 meq kg)1, which was lower than 27.6 meq kg)1 reported for fresh sardine by Cho et al (1989) During storage period, the sardine with rosemary extract showed generally low lipid oxidation compared to the control without rosemary extract as reported for other fish species (Vareltzis et al., 1997; Gimenez et al., 2004, 2005; Da Silva Afonso & Santana, 2008; Sarkardei & Howell, 2008; Tironi et al., 2009) TBA is second breakdown product of lipid oxidation and widely used as an indicator of degree of lipid oxidation The concentration of TBA in freshly caught fish is typically between and mg of malondialdehyde (MDA) equivalents per kilogram flesh, but levels of 5–8 mg of MDA equivalents per kilogram of flesh are generally regarded as the limit of acceptability for fish stored in ice (Nunes et al., 1992) Table also shows TBA contents in the different treatments during storage TBA values indicating rancidity development in the all fish flesh remained low ( 0.05) in FFA concentrations were observed between the treatment groups (R1 and R2) throughout storage period Although the release of FFA increased from the initial value of 2.88 (expressed as percentage of oleic acid) to the final value of 7.23 for the control, 5.98 for R1 and 6.13 for R2 at the end of storage period, lipid hydrolysis developed at a Ó 2010 The Authors Journal compilation Ó 2010 Institute of Food Science and Technology Table Changes in the value of free fatty acids during storage period Control Storage days 10 13 17 20 R1 X Ỉ Sx 2.88 2.79 3.11 3.40 3.39 6.67 7.23 ± ± ± ± ± ± ± R2 X Æ Sx a 0.96 0.22a 0.37a 1.10a 0.61a 0.19b 0.43a 2.88 2.65 2.83 3.37 3.97 5.34 5.98 ± ± ± ± ± ± ± X Ỉ Sx a 0.96 0.26a 0.51a 0.27a 1.82a 0.29a 1.00a 2.88 3.03 3.37 3.60 3.10 5.09 6.13 ± ± ± ± ± ± ± 0.96a 0.32a 0.07a 0.26a 0.22a 0.39a 0.90a Different letters in the same row indicate significant differences (P < 0.05) X Ỉ Sx , average ± standard deviation; C, control; R1, 1% rosemary; R2, 2% rosemary International Journal of Food Science and Technology 2010 1721 Rosemary extract in preventing oxidation Y Ozogul et al Conclusion TVC content of sardine in VP 12 10 Log CFU g–1 1722 Control R1 R2 0 10 13 Storage days 17 20 Figure Total viable count (TVC) of sardine in VP at °C slower rate in the samples treated with rosemary extract, regardless of the level of antioxidant Microbiological assessment TVC are used as an acceptability index for fish products because of the effect of bacteria in spoilage TVC determined in sardine fillets were initially 4.22 log CFU g)1, which was higher than those reported for sardine (El Marrakchi et al., 1990; Oăzogul et al., 2004) Comparison with the proposed limits (5–7 log CFU g)1) for fresh fish (ICMSF, 1986) shows that sardine fillets were of good quality TVC increased with storage time for all groups (Fig 1), and the growth of microorganisms exceeded the limit on day 10 for the control, on day 13 for R1 and on day 17 for R2 It can be concluded that the shelf life of sardine in VP was approximately 8–9 days for the control, 11–12 days for R1 and 13–14 for R2, indicating that sensory analysis did not correlated well with microbiological analysis Lemon extract and thymol in combination with MAP significantly (P < 0.05) reduced the growth rate of bacterial population compared with the control (Del Nobile et al., 2009) In this study, changes in TVC during storage also showed the existence of a reduced growth in the samples with rosemary extract The result obtained from sensory evaluation, after rosemary treatment, showed a longer shelf life when compared with microbiological results The spoilage rate of fish depends on the species and type, the initial microbial flora, the location of the catch, the content of the catch, processing methods and method of storage After being caught and handled, fish are exposed to additional contamination on board fishing vessels Unhygienic practices, especially unwashed hands, clothes, equipment, decks and storage facilities, can contaminate fish To avoid contamination of fish, all equipment must be cleaned and sanitized International Journal of Food Science and Technology 2010 Based primarily on sensory assessment, vacuum-packed sardine fillets (C, R1 and R2) reached the limits of acceptance 13, 17 and 20 days of storage, 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(2009) Lipid and protein changes in chilled sea salmon (Pseudopercis semifasciata): effect of previous rosemary extract (Rossmarinus officinalis L.) application International Journal of Food Science & Technology, 44, 1254–1262 Vareltzis, K., Koufidis, D., Gavriilidou, E., Papavergou, E & Vasiliadour, S (1997) Effectiveness of a natural Rosemary (Rosmarinus officinalis) extract on the stability of filleted and minced fish during frozen storage Zeitschrift fur Lebensmittel –Untersuchung und -Forschung A, 205, 93–96 International Journal of Food Science and Technology 2010 1723 ... on the effects of different level of rosemary extract on the quality of vacuum-packed sardine stored at °C The principal objectives of this investigation were (i) to determine the shelf life of the. .. Effects of pretreatment with rosemary (Rosmarinus officinalis) in the prevention of lipid Ó 2010 The Authors Journal compilation Ó 2010 Institute of Food Science and Technology Rosemary extract in preventing... and VP prolongs the shelf life of sardine to a minimum of days, depending on the concentrations of rosemary extract Oxygen availability is the most critical factor in the development of lipid