Application of Green Bio-Preservatives in Extending the Shelf Life of      Commercially Important Fishes Sardinella Longiceps and Rastrelliger      Kanagurta

INTRODUCTION

Fish, the primary dietary source [1-3] with high protein and omega-3 fatty acid [4] is well known for its protective and anti-inflammatory properties since ancient times. But the shelf‐life of such prime seafood is limited because of its high contents of various nutrients, neutral pH, high moisture content, etc. According to WHO, half of the seafood consumed by the consumers is fresh and live fish. Moreover, the consumers are very keen on the freshness which is associated with safety, reassurance, and superior taste. Anvari et al. (2013) stated that 70% of the total fish catch in developing countries were preserved using various basic food preservation techniques like freezing, chemical preservation, salting, and smoking to improve the microbial safety and increase the shelf life of fish and shellfish [5]. But Stolyhow et al. (2006) reported that these basic techniques not only increased the lipid oxidation but also decreased the nutritional quality of the fish [6]. So, synthetic preservatives were widely used in fish storage to extend shelf life and maintain quality and safety to prevent the nutritional and sensory losses caused by microbiological, enzymatic, or chemical changes, and shelf‐life extension of food are usually achieved so far by chemical preservatives, such as sodium benzoates, sodium nitrite, and sulfur-di-oxide. Nonetheless, the accumulation of these synthetic preservatives in tissues can be detrimental to health. However, the use of these preservatives has been linked to potential health hazards. In this regard, natural preservatives with excellent antioxidant and antimicrobial properties have been extensively searched and implemented as safe alternatives in seafood processing, with the sole purpose of extending shelf‐life. Natural preservatives from microorganisms, plants, and animals have been shown potential in replacing the chemical and synthetic antimicrobials. Plant extracts have been recently received considerable attention in seafood processing as alternatives for synthetic preservatives [7, 8].

Pragmatically loss of freshness of fish is the consequence of post-mortem biochemical, physicochemical, and microbiological processes as well as several extrinsic factors such as the handling onboard and on land, and technological processing for which natural antioxidants have been generally considered safe for human consumption. The potential risk to human health on consuming frozen seafood has not been adequately investigated. With the view to help humans to overcome malnutrition problems and fish protein demand, the present study was undertaken with the objectives to assess the effect of frozen storage period - a week i.e. 0-7 days - at (4±1ºC) temperature on the fish fillet of selected marine fishes with and without preservation.

MATERIALS AND METHODS

Collection and preparation of preservative paste: The collected and identified Sargassum wightii from Mandapam (9°16'48" N: 79°07'12" E), Rameswaram coast, Coleus aromaticus from a local farm were washed thoroughly using tap water. Later the cleaned materials were cut into small pieces, shade dried for two weeks, powdered by grinding them in a lab electric mixer grinder, and stored in a refrigerator. Whenever required, 100 gm of collected materials were ground for 2-5 mins (each separately) with adding water as a solvent and the paste was used for further analysis.

Storage Study: Selected experimental fishes Sardinella longiceps (EF1) and Rastrelliger kanagurta (EF2) of little size were purchased from the landing center and fish market of Olavakkodu, Kerala, and fillets were made with a mean weight of 100-150 g. Then, the fillets were covered with a paste made from macroalga Sargassum wightii and natural Coleus aromaticus leaves, the fillets were placed in sterile polythene bags and stored at chilled temperature (4±1 ºC). After 24hrs, the samples in triplicates were randomly removed from the treatment to evaluate the preservative of macroalga extracts on Sardinella longiceps and Rastrelliger kanagurta fillets. The fish fillets were stored with and without preservatives in the refrigerator at 4±1°C for a week (1^st, 3^rd, 5^th, and 7^th day) of the preservation period and analyzed for the following parameters.

Wet lab analysis

Proximate composition: For fresh and preserved fish, the proximate composition was determined from the body muscle. Following parameters like ash and moisture of preserved and unpreserved fish samples were evaluated using standard methods such as AOAC (2000) [9]. The protein content was determined by the methods described by Lowry et al. (1951) [10]. The total carbohydrate was determined using Hedge and Hofreiter methods (1962) [11]. Fat content was determined using Floch et al. (1957) [12]. 

Sensory Evaluation: The sensory evaluation was carried out by a group of panelists according to the method described by Potter (1968) [13]. A sensory panel was formed consisting of twelve experienced judges (6 males and 6 females; 30-55 years old). The steam-cooked samples of fresh and preserved fish were evaluated for quality attributes including texture, flavor, color, appearance, general taste, and overall acceptability by using 5-point hedonic scales (0 = excellent; 1= very good; 2= good; 3-4 fair; <5 bad) and score 5.0 was considered the borderline of fish acceptability.

Microbial Analysis: 10 g of fish sample was weighed aseptically and homogenized with 90 mL of physiological saline solution. Appropriate dilutions were made from the 9.0 mL physiological saline and plated onto the potato dextrose agar plate containing antibiotics or tartaric acid solution. The plates were incubated at room temperature for four days and all colonies were counted and the data was reported as CFU g^-1.

Statistical analysis: Data were analyzed using SPSS (Scientific Package of Social Science) version 16.0. The mean, standard deviation (SD), and one-way ANOVA test were performed to compare differences in parameters analyzed among selected fish species and a test for the comparison of means (P < 0.05).

RESULTS AND DISCUSSION

Proximate composition

Ash: Result shown in Table 1 revealed that the ash content decreased significantly from 2.28±0.01 to 1.94±0.01 in EF1 similarly 2.29±0.02 to 2.05±0.01 in EF2 on 0^th day to 7^th day of untreated frozen storage at 4±1ºC. There was a decrease of 2.25±0.01 to 2.10±0.04 in EF1 and an increment of 2.75±0.21 to 4.05±0.08 in EF2 on 0^th day to 7^th day preserved with Coleus aromaticus (Table 2). Table 3 showed that initially on day 0 ash content was 2.20±0.03 and it decreased significantly to the value 1.89±0.09 in EF1 and 2.53±2.43 to 2.55±0.15 in EF2 on 0^th day to 7^th day of storage at 4±1ºC preserved with Sargassum wightii (Table 3). The increase in ash contents of fish products during frozen storage might be attributed to the loss recorded in the concentration of protein and fat content, which reflected the increasing, found in ash contents. A decrease in ash content of fish during frozen storage was attributed to the drip loss during the thawing process [14]. The present results of ash content are in agreement with Beklevik et al., (2005) on sea bass fillets.

Moisture: In the initial day of storage (Day-0) of fish muscle without a preservative, the moisture content (% of wet basis) recorded in EF-1 was 72.02±0.05 and 73.02±.09 in EF-2, while in fish muscle preserved with preservative Coleus aromaticus it was 74.40±0.11 (EF1), 77.57±0.27 (EF2) and with preservative Sargassum wightii it was 73.23±0.15 (EF1) and 74.25±0.18 (EF2). At the end of the storage period (i.e 7^th day), the moisture content still got decreased in Coleus aromaticus treated fish tissue to 70.5±0.18 in EF1 and 81.05±0.57 in EF2 and Sargassum wightii treated fish tissue to 68.14±0.33 in EF-1 and 69.15±0.33 in EF-2. The moisture content of the ice stored fish increased from the initial value till 3rd day which could be due to the absorption of melted ice water by the fish muscle. The later gradual decrease was observed on the 7th day. It might be due to the evaporation of moisture from the surface, which relies on various factors like geometric shape, the chemical composition of the product, storage temperature, and relative humidity, and these findings were supported by Dhanapal et al. (2013) [15].

Protein: Perusals of table 1 revealed that protein content was reduced significantly from 0^th day (19.15±0.15) to 7^th day (13.05±0.15) in EF-1 and 21.32±0.18 to 14.02±0.17 in EF-2 of untreated fish muscles stored at 4±1ºC. A significant percent increase (P˂0.05) was found in EF-1 and EF-2 preserved with Coleus aromaticus from 0^th day 25.44±0.16 (EF1) and 28.44±0.05 (EF2) to 7^th day 39.24±0.20 (EF1) and 40.00±0.93 (EF2) of storage at 4±1ºC (Table 2). Table 3 showed that initially on day 0 protein content was 27.34±1.10 and it increased significantly to the value 45.23±1.21 in EF1 and 28.35±1.15 to 46.32±1.26 in EF2 on 7^th day of storage at 4±1ºC preserved with Sargassum wightii. The decrease in protein is due to leaching effect of amino acids and water-soluble protein leaching out with melting ice attributed by early researchers [14, 16-19]. These values showed that the two fish species EF-1 and EF-2 preserved with preservatives contained high levels of proteins at the end of Day 7 which can be due to the impact of crude protein of preservatives so it can be used as a preservative on animal protein sources [20, 21].

Lipid: The results in the table 1 revealed that the lipid content decreased significantly (P˂0.05) from 0^th day 3.01±0.14 (EF1) and 3.52±0.17 (EF2) to 7^th day 2.21±0.01 (EF1) and 2.48±0.07 (EF2) of untreated fish muscle stored at 4±1ºC. There was an increase in lipid content of fish muscle treated with Coleus aromaticus from 0^th day 2.08±0.11 (EF1) and 2.28±0.32 (EF2) to 7^th day 2.27±0.09 (EF1) and 2.95±0.82 (EF2). Table 3 showed that initially on day 0 lipid content was found to be 2.05±0.14 and it increased significantly to the value 2.22±0.06 in EF1 and 2.22±0.17 to 2.43±0.05 in EF2 on the 7^th day of storage at 4±1ºC preserved with Sargassum wightii. Rasoarahona et al. (2005) reported that the lipid content of fish differed which could be due to variation of species, diet, geographical origin, age, and season, which supported the present report on lipid content in EF-1 and EF-2 [22].

Carbohydrate: During the study period on the initial day of storage (Day 0) of the fish muscle without preservative, the amount of carbohydrate content (% of wet basis) recorded in EF-1 was 0.41±0.01 and 0.52±0.04 in EF-2; while in fish muscle preserved  with  preservative  Coleus

Table 1. Proximate chemical composition of marine fishes EF-1 and EF-2 muscle samples (without preservative) under chilled storage condition (4 ±1°C) with respect to storage period.

*Each value is the mean ± SE of three replicates. Mean values followed by different letters in the same column were significantly different (P<0.05)

Table 2. Proximate chemical composition of marine fishes EF1 and EF2 muscle samples (with preservative-1) under chilled storage condition (4 ±1°C) with respect to the storage period.

*Each value is the mean ± SE of three replicates. Mean values followed by different letters in the same column were significantly different (P<0.05)

Table 3. Proximate chemical composition of marine fishes EF1 and EF2 muscle samples (with preservative-2) under chilled storage condition (4 ±1°C) with respect to the storage period.

*Each value is the mean ± SE of three replicates. Mean values followed by different letters in the same column were significantly different (P<0.05)

aromaticus was 0.65±0.02, and preserved with Sargassum wightii was 0.30±0.06 in EF-1. In EF-2, it was 0.52±0.02 for Coleus aromaticus and 0.45±0.08 for Sargassum wightii. An increase in the value of carbohydrate content (0.93±0.10

and 0.55±0.48) was noted in the fish muscle of EF-1 and EF-2 preserved with Coleus aromaticus on 7^th day of storage. Similar trend was observed in Sargassum wightii in EF-1 (0.82±0.21) EF-2 and (0.92±0.21).

Table-4. The changes in total viable counts (TVCs) of fish muscle samples during chilling storage (4±1ºC) with respect to storage period.

Table 5. The quality index score of the selected commercial important marine fishes Sardinella longiceps (EF-1 ) and Rastrelliger kanagurta (EF-2) collected from Olavakkode Fishmarket.

Note: * 0-Excellent; 1-Very good; 2-Good; 3-Moderate; 4-Bad; 5-unacceptable