Background: food industries use synthetic preservatives to improve the quality and enhance the shelf life of food products during storage. However, the most common industrial strategies for preservation may not always bring the desired protection for curbing corruption. Furthermore, consumer demands for safer foods encourage researchers to find natural and effective preservatives. In this study, the antioxidant and antibacterial activity of chitosan in combination with cinnamon were investigated in frozen condition. Methods: rainbow trouts were combined with 2% chitosan in combination with different concentration of cinnamon essential oils (0.125, 0.25 and 0.5 mM). The samples were kept at -18 ºC. Oxidative stability of samples was assayed by measuring lipid peroxidation level using thiobarbituric acid reactive substances (TBARS) method. The bacterial test was assayed by counting colony forming unit. Results: combination of chitosan with 0.25 and 0.5 mM cinnamon significantly decreased lipid peroxidation level compared to control group and chitosan combination with 0.5 mM cinnamon showed synergist effect. The antimicrobial activity of chitosan in combination with 0.5 mM cinnamon was higher than other concentrations and control groups. Conclusion: chitosan in combination with cinnamon oil could considerably increase the oxidative stability and decrease total count of bacteria in frozen fish. These results may suggest that these edible coatings can be used instead of artificial preservatives and non-edible coatings.
Keywords: chitosan, cinnamon, lipid peroxidation, antibacterial, antioxidant
Freezing is a general preservation method used to control or decrease biochemical changes that occur during storage in fish determined by chemical indicators of spoilage such as Thiobarbituric acid (TBA) values, Total Volatile Base Nitrogen (TVB-N) values and the pH values (14). Nevertheless, frozen storage does not completely inhibit chemical reactions (e.g., lipid oxidation) that lead to quality deterioration of fish tissue. Food industries commonly used antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) to improve the quality and increase the shelf life of food products during storage. Numerous studies have currently focused on using natural ingredients to enhance food quality and shelf life to meet consumer demands for safer foods in order to avoid the use of synthetic preservatives 1.
The chitosan, the second most abundant natural polymer after cellulose, is used in food stuff, agriculture, cosmetic, pharmaceutical industries and waste water treatment 2. Chitosan extracts from waste of shrimp, about 50 percent of total body weight of shrimp is waste 3. Antioxidant activity is one of the well-known functions of chitosan. Several mechanisms about the antioxidant action of chitosan have been proposed (1). One of the features of chitosan is antimicrobial activity. The degree of this activity depends on the methods involved in the production of chitosan (3). In order to control undesirable microorganisms in foods, antimicrobial substances can be incorporated in edible films (4). Furthermore, many spices and herbs possess antimicrobial and antioxidant activity, which minimizes questions regarding their safety in food products. Usually, compounds with phenolic groups are most effective. Among these, the oils of clove, thyme, cinnamon, rosemary, sage and vanillin have been found to be effective against microorganisms. These compounds have more inhibitory effects against gram-positive bacteria in comparison with gram-negatives (10).
Current work was therefore carried out to investigate the quality of frozen fish with edible coating of chitosan in combination with cinnamon after 40 days storage.
2. Materials and methods
2.1. Preparation and treatments of fish samples
Rainbow trout fish were prepared from fish farm with an average weight of 700 gr and the average length of 30 cm. The fillets were prepared with 25 g weight. The fish fillets were divided into three treated groups and one control group. Cinnamon (Givaudan, Switzerland) solutions were prepared at concentrations of 0.5, 0.25 and 0.125 mM in the solution of 2% of chitosan (2 g chitosan was dissolved in 100 ml of glacial acetic acid).
For mixing chitosan with different concentrations of cinnamon essential oil, 0.5 ml of chitosan was added to 0.25 ml of cinnamon essential oil for every gram of meat fillet. The samples were randomly assigned into four treatment lots, one control group without chitosan and cinnamon and three groups with 2% chitosan and different concentrations of cinnamon (0.125, 0.25 and 0.5 mM). Then the fish fillets was dried and stored at -18 °C for 40 days. .
2.2. Bacterial test
At first 25 g of meat was homogenized with 225 ml of distilled water. We tried to make further dilutions up to 10-7. In order to culture and measure total count of bacteria, 1 cc of 10-1 to 10-7 dilutions was poured into sterile nutrient agar medium plates in sterile conditions and was incubated at 37 ° C for 24-48 h. Afterward, the entire colony that grew on the surface was counted regardless of the detection of species and was reported as cfu/g.
2.3. Assay of chitosan and cinnamon power on the oxidative stability in sunflower oil
Malodialdehyde (MDA) levels, as an index of lipid peroxidation, were measured. MDA reacts with thiobarbituric acid (TBA) as a thiobarbituric acid reactive substance (TBARS) to produce a red colored complex which has peak absorbance at 532 nm. The chitosan and cinnamon, Copper sulfate (CuSo4), sunflower oil (without antioxidant) were mixed and shaked for 3 h in room temperature. The capacity of the chitosan and cinnamon to inhibit MDA formation against CuSo4 was assayed. Briefly, the samples were mixed with 20% trichloroacetic acid and the mixture was centrifuged. Then, thiobarbituric acid was added to the supernatant and heated. The absorbance of the supernatant was measured at 532 nm. The values were expressed in nM of malodialdehyde, using a molar extinction coefficient of 1.56 × 105 M-1 cm-1(5).
3.1. Antimicrobial Properties of chitosan and cinnamon
Total count was measured after 20 and 40 days from freezing. Our results indicated that chitosan in combination with different concentration of cinnamon significantly decreased total count of bacteria in comparison with the control group after 20 and 40 days from freezing (p < 0.001, table 1).
3.2. Antioxidant activity of chitosan and cinnamon
The results of antioxidant activity are given in table 2. This table shows results of the efficacy of chitosan in combination with cinnamon on the shelf life of frozen rainbow trout in the period of 40 days.
In the first stage (after 20 days), there was significant difference between the oxidative stability of control sample and chitosan in combination with cinnamon essential oils at concentrations of 0.25 and 0.5 mM (p < 0.05) but there was no significant difference between the oxidative stability of control sample and chitosan in combination with cinnamon essential oils at 0.125 mM concentration.
Also after 40 days, a significant difference was shown between the oxidative stability of control sample and chitosan in combination with cinnamon essential oils at 0.25 and 0.5 mM concentrations (p < 0.01 and p < 0.001, respectively) but there wasn't significant difference between the oxidative stability of control sample and chitosan in combination with cinnamon essential oils at 0.125 mM concentration.
Furthermore, there was a significant difference between the control sample in the first stage and the control sample after 40 days (p < 0.001).
The present study investigated the shelf life of frozen rainbow trout in the presence of chitosan in combination with cinnamon essential oils in the period of 40 days.
The obtained results revealed that in both control samples and chitosan in combination with cinnamon essential oils, lipid peroxidation increased from the first to the second stage, so during storage lipid oxidation definitely had an ascending trend.
The level of peroxidation in treated samples was less than the control sample. It indicates that chitosan in combination with cinnamon essential oils provide good oxidative stability in frozen fish. Therefore, treated samples have more antioxidant components than control sample.
Fish and other types of marine-derived foods have been recognized as valuable sources of high nutritional components. These types of food are key ingredient in many countries’ diets. They are good sources of long-chain polyunsaturated fatty acids (PUFAs) belonging to the Omega-3 family, including EPA (20:5n3) and DHA (22:6n3). DHA in the diet has a positive effect on preventing and curing several diseases such as coronary heart disease, atherosclerosis and some cancers (10).
PUFAs are vulnerable to free radical damages and oxidation. In the current study, considerable change in the level of lipid oxidation was evaluated between 20 days and 40 days of storage in control groups. Presence of antioxidants plays an important role in preventing oxidative changes of these valuable components.
Regarding previous reports investigating the oxidative stability of frozen shrimp in the presence of carotenoids and chitosan, both chitosan and carotenoids coating are effective for preserving frozen shrimp during refrigerated storage.
Gram-negative psychotropic bacteria are the major group of microorganisms responsible for spoilage of aerobically stored fresh fish at chilled temperatures (11).
The total count of bacteria increases during storage. It has been shown that chitosan alone and in combination with cinnamon essential oils has antibacterial effect 2. The obtained results from our study revealed that chitosan in combination with cinnamon essential oils could decrease the level of microbial count in the samples stored at -18°C after 20 days. After 40 days, total count was measured again. The microbial load had sharply dropped that it would be appropriate because of the coating of chitosan in combination with cinnamon as well as freezing conditions.
After 40 days, our data showed that 0.5 mM concentration of cinnamon essential oils and chitosan were more effective to reduce total count of bacteria compared to other samples and control group.
Recent studies have shown that chitosan and cinnamon essential oils have synergistic effects in decreasing the total viable count 4. Therefore, coating of chitosan in combination with cinnamon oil provides an active type of coating that can be utilised as a safe preservative for fish under refrigerated storage 3. Previous studies have also identified trans-cinnamaldehyde as the major antibacterial constituent of cinnamon oil (12).
The antimicrobial properties of chitosan coating have been reported in the literature (13). S.M. Ojagh et al investigated the effects of a chitosan coating enriched with cinnamon oil on quality of rainbow trout (Oncorhynchus mykiss) during refrigerated storage, over a period of 16 days. They reported that chitosan in combination with cinnamon oil coating is able to retain longer and extend the shelf life of fish samples during the refrigerated storage (11).
Our study showed that chitosan in combination with cinnamon could reduce total count of bacteria in freezing conditions after 40-days period and 0.5 mM cinnamon had the greatest influence.
Current results revealed that coating of chitosan in combination with cinnamon oil can decrease the level of lipid peroxidation as well as total count of bacteria in frozen fish leading to extend the shelf life of fish samples. These results may suggest that this edible coating can be used instead of artificial preservatives and non-edible coatings.