Abstract: Inhibition efforts for quality deterioration in fish can be performed using any preservatives. Preservatives that used must be sourced from natural ingredients, in order to minimize the effects of hazardous chemicals when using synthetic materials. One of natural preservatives from marine is the extract of Gracilaria sp. The fresh red tilapia were prepared to form the fillets and then soaked for 30 min in Gracilaria sp. extract solution with a concentration of 0, 0.5, 1, 1.5 and 2% then drained for 15 min. Red tilapia fillets stored at low temperature (6°C) for 10 days with an interval of observation every 2 days. Observations made on the organoleptic, measurement of pH, Total Volatile Bases (TVB) and Total Plate Count (TPC). Based on the value of pH, TVB and TPC, the extract of Gracilaria sp. it enabled to extend the storage life of the fillet from 6-10th day. Based on organoleptic, extract of Gracilaria sp. enabled to maintain the parameters of appearance, texture and smell of red tilapia fillet from 4-6th day of storage. The use of extracts of Gracilaria sp. 2% gave the best results based on the parameters of odor, pH, TVB and TPC.
INTRODUCTION
Fresh fish (including red tilapia) are more susceptible compared to red meats and chicken and its quality decreases due to a complex process in which physical, chemical and microbiological forms of deterioration are implicated (Gonzalez-Fandos et al., 2005). Enzymatic and chemical reactions are responsible for the initial loss of freshness whereas microbial activity is responsible for spoilage (Gram and Huss, 1996). Therefore, it needs to be done attempt to increase the shelf life of fishery products through processing and preservation, one of which is low-temperature storage. Low-temperature storage besides to inhibiting the activity of microbes and enzymes also can retain the original properties of the fresh fish. However, storage of fresh fish at low temperatures still have a limited shelf life (Santoso et al., 1999). Therefore we need safe of preservatives in the product in order to have a good quality with a longer shelf life.
Seaweed is one of the many marine biota known to contain active compounds that are useful as antibacterial, antivirus and antitumor (Atmadja, 1992). According to Husni (2006), Gracilaria sp. is one of macroalgae that has potential as antibacterial. This type of marine algae are found on the south coast of Yogyakarta. This study aimed to determine the effect of extracts of Gracilaria sp. as preservatives of red tilapia fillet stored at refrigerator.
MATERIALS AND METHODS
Materials: Red tilapia (size±250 g fish-1) were obtained from freshwater fish of Mina Kepis Sleman Yogyakarta. Gracilaria sp. obtained from the Drini beach Gunungkidul Yogyakarta. Staphylococcus aureus obtained from the Laboratory of Agricultural Microbiology, Faculty of Agriculture, University of Gadjah Mada and Pseudomonas aeruginosa from the Laboratory of Food Microbiology, University Centre of the University of Gadjah Mada. Trypticase Soya Broth (TSB), Tryptone Soya Agar (TSA), TCA, K2CO3 obtained from Difco (Detroit, MI, USA).
Extraction procedures: Extraction Gracilaria sp. using the method by Husni (2006) with modifications. A total of 750 g of Gracilaria sp. washed with clean water and then rinsed using PBS and then cut into pieces and added to 96% ethanol (1:4) further blended for 30 min. Extracts have been obtained is evaporated using a rotary evaporator with a temperature of 40°C. Further drying process by using a freeze dryer.
Antibacterial activity test: The test antibacterial activity of extracts of Gracilaria sp. against S. aureus and P. aeruginosa used Kirby-Bauer method as described by the Fadhlan (2010).
The use of extract Gracilaria sp. on red tilapia fillet: Preparation of red tilapia fillet using the method described by NSA (2006a). The use of extracts of Gracilaria sp. i.e., by tilapia fillet soaked in the extract solution. Extract solution made by adding powdered extract Gracilaria sp. into distilled water at various concentration of 0, 0.5, 1, 1.5 and 2%. Fillet was prepared then immersed into the extract for 30 min at room temperature. After soaking for 30 min then drained and then put in the refrigerator with low temperature (6°C) for 10 days. Each treatment was repeated 3 times. Testing parameters include: pH (AOAC, 1990), Total Plate Count (TPC) (NSA, 2006a), Total Volatile Base (TVB) (AOAC, 1995) and sensory analysis (NSA, 2006b).
Statistical analysis: SPSS for Windows (version 11.0, SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All data were expressed as Mean±SD (SD). The significance of differences (p<0.05) among the corresponding mean values was determined by using one-way analysis of variance (ANOVA) followed by Duncan’s new multiple-range test.
RESULTS AND DISCUSSION
Antibacterial activity: The in vitro antimicrobial activities of Gracilaria sp. extracts against Staphylococcus aureus and Pseudomonas aeruginosa were qualitatively and quantitatively assessed by the zone diameters and MIC values. The results were given in Table 1 show that Gracilaria sp. from the Drini beach Gunungkidul Yogyakarta has antibacterial activity against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) which is higher antibacterial activity on S. aureus than P. aeruginosa. The maximum inhibition zones and MIC values of Gracilaria sp. extract were 13.7 mm and 30 mg mL-1 for S. aureus and 8.4 mm and 300 mg mL-1 for P. aeruginosa, respectively. Research conducted Maduriana and Sudira (2009) showed that Gracilaria arcuata from Batu Bolong beach Canggu Bali and Gracilaria lichenoides from the coast of Serangan Bali also have higher antibacterial activity against Gram-positive (Micrococcus luteus) than Gram-negative (E. coli). According Pelczar and Chan (1977) the growth of Gram-positive bacteria more easily inhibited than Gram-negative bacteria.
| Table 1: | Inhibition activity of extract of Gracilaria sp. against S. aureus dan P. aeruginosa |
Weaker antibacterial activity in P. aeruginosa is possible because the active compounds from the extract of Gracilaria sp. rather difficult to penetrate the bacterial cell wall of P. aeruginosa. Gram-negative bacteria have an outer membrane that protects bacteria from toxic substances (Prescott et al., 1993). Outer membrane has narrow pores that adds protection for Gram-negative bacteria. Outer membrane in Gram-negative bacteria may hinder antibacterial, dyes and detergents into the inside of the bacterial cell. The different in the composition and structure of the bacterial cell wall of Gram-positive and Gram-negative bacteria cause different responses in the two groups of bacteria (Prescott et al., 1993).
P. aeruginosa is a Gram-negative bacteria that have a cell wall structure is more complex and contains more lipid components (11-12%) compared to the structure of the cell wall in bacteria S. aureus. Thus, the cell wall of bacteria S. aureus will be more easily damaged by bioactive compounds contained in the extract of Gracilaria sp. Inhibition of cell wall synthesis would lead to the bacterial cell wall is weakened and becomes lysis. Lysis of cells caused by cell wall no longer function in maintaining the shape and protect bacteria that have a high osmotic pressure. In addition, the bacteria S. aureus has an osmotic pressure in the cells 3-5 times larger than Gram-negative bacteria, making it more susceptible to lysis (Jawetz et al., 2001).
Degree of acidity (pH): The changes in the pH of red tilapia fillet were given extracts of Gracilaria sp. during stored at cold temperatures are described in Fig. 1. The initial pH of the fish samples was 6.6. The pH of the samples decreased initially and then increased. The initial pH decrease may be attributed to the dissolution of CO2 in the fish samples. Several authors have reported a decrease in the pH of fish samples with increase in the concentration of CO2 in the atmosphere (Manju et al., 2007; Lannelongue et al., 1982).
| Fig. 1: | Effect of Gracilaria sp. on pH of red tilapia fillet during storage at low temperature |
The increase of pH may be caused to the production of volatile basic components, such as ammonia and trimethylamine by fish spoilage bacteria (Ruiz-Capillas and Moral, 2001).
The results of the analysis showed that the pH occurred significant difference between treatments after 8 and 10 days, were the 0-6 days there is no significant difference and all the fillet still in good condition. At the end of the observation (10 days) showed treatment of extract of 2% has a pH of 6.70 which means by treatment with 2% red tilapia fillet able to maintain the pH within the threshold of fresh fish while other treatments showed pH values above the threshold pH of fresh fish.
Total volatile base (TVB): Total Volatile Base (TVB) which is mainly composed of ammonia and primary, secondary and tertiary amines, resulted from degradation of proteins and non-protein nitrogenous compound which is caused by microbial activity (Ruiz-Capillas and Moral, 2005). The TVB content of red tilapia fillet during storage indicates that the value is always increasing (Fig. 2). Purwaningsih et al. (2005) said that TVB changes can be caused by temperature and length of storage time. The increase in TVB red tilapia fillet with treatment of extract of Gracilaria sp. relatively small compared to the control (0%). This is because of the antibacterial compounds in extracts of Gracilaria sp. capable of inhibiting bacterial decomposition of protein components into a volatile compound. Suptijah et al. (2008) suggested that the increased value of TVB in fish during storage due to degradation of the protein or its derivatives to produce a range of volatile base such as ammonia, histamine, hydrogen sulfide and trimethylamine.
TVB content on the first day of observation does not happen real difference. This is because red tilapia fillet is still in a state of fresh and TVB content is very low (4.2 mgN/100 g).
| Fig. 2: | Effect of Gracilaria sp. on TVB of red tilapia fillet during storage at low temperature |
At the end of the observation, the extract of 2% has the lowest value of TVB (21.64 gN/100 g). According to Connell (1995) when the content of TVB in fish above 30 g gN/100 an indicator of decaying fish. Extract Gracilaria sp. 2% are able to maintain the tilapia fillet 4 days longer than without the extract, for treatment without extract (0%) fillet shelf life only until 6 days.
Total plate count (TPC): Figure 3 shows that the total bacteria on red tilapia fillet increased along with the length of storage, but the number of bacteria on a red tilapia fillet were given the extract Gracilaria sp. 2% lower than other treatments. It shows that the use of extract of Gracilaria sp. can inhibit the activity of bacteria on red tilapia fillet. The increase in total bacteria on red tilapia because fish fillet is a suitable medium for bacterial growth.
The maximum limit for total bacterial fish fillet products is 5.69 log CFU g-1 (NSA, 2006c). After 6 days, total bacteria in all treatments still in the acceptable threshold, but after 8 days the red tilapia fillet with the addition of extract 0 and 0.5% have exceeded the maximum number of total bacteria. Extract with a concentration of 2% showed the lowest TPC during storage and until the end of the observation showed a concentration of 2% had 5.40 log CFU g-1 and below of maximum allowable TPC. Thus it can be said that the best treatment is to give the extract at a concentration of 2% because it is able to inhibit the growth of bacteria until day 10. According to Fenical and Paul (1984), red algae contain compounds terpenoids, acetogenin and halogen compounds are a group of compounds that function as plant antibakteri. Terpenoid have significant benefits as a traditional medicine, antifungal and antibacterial (Bohlmann et al., 1998).
Sensory analysis: The acceptability of fish and fishery product during cold temperature storage depends on the changes in their sensory attributes including appearance, texture and odor.
| Fig. 3: | Effect of Gracilaria sp. on TPC of red tilapia fillet during storage at low temperature |
| Table 2: | Effect of Gracilaria sp. on score of appearance of red Tilapia fillet during storage at low temperature |
| Values in the same column with different letters are significantly different at p<0.05 | |
| Table 3: | Effect of Gracilaria sp. on score of texture of red Tilapia fillet during storage at low temperature |
| Values in the same column with different letters are significantly different at p<0.05 | |
Based on Table 2 it can be seen that the appearance of red tilapia fillet after 6 days who were given the extract with a concentration of 1.5 and 2% had a good appearance score (more than 7). According to INS-01-2346-2006, fish is still fresh when the sensory test value ranges between 9-7 and not fresh when the value of sensory tests ranged from 4-1 (NSA, 2006c). Wicaksono (2010) reported that the addition of extracts of Sargassum sp. 1% in the red tilapia fillet is the best treatment in maintaining the highest organoleptic values for red tilapia fillet appearance parameters are stored at low temperatures. Wibowo (1993) suggested that the extract Sargassum sp. 2% is added to the mackerel fillet is the best treatment to preserve the value of the highest organoleptic appearance mackerel fillet during storage in ice.
Table 3 shows that treatment with a concentration of 1.5 and 2% had a good texture score (more than 7) after 6 days, whereas treatment without the extract (0%), 0.5 and 1% only until 4 days. Wicaksono (2010) reported that the extract of Sargassum sp. 1% is added to the red tilapia fillet is the best treatment to preserve the highest value of texture red tilapia fillet stored at low temperatures.
Table 4 shows that the storage up to 4 days, the odor of all red tilapia fillet still within the limits of acceptance, but after the 6 days of treatment only with a concentration of 2% which indicates an acceptable odor score (7.1) while for other treatments have scores odor below 7. Wicaksono (2010) reported that the extract of Sargassum sp. 1% is added to the red tilapia fillet is the best treatment in maintaining the highest organoleptic values for red tilapia fillet were stored at low temperatures.
| Table 4: | Effect of Gracilaria sp. on score of odor of red Tilapia fillet during storage at low temperature |
| Values in the same column with different letters are significantly different at p<0.05 | |
Wibowo (1993) mentions that the extract Sargassum sp. 2% was added to the mackerel fillet is the best treatment to preserve the highest value for the parameter organoleptic odor mackerel fillet during storage in ice. The factors that cause the difference in odor scores for each treatment because of the difference of extract of Gracilaria sp. Extract Gracilaria sp. contain antibacterial that can inhibit the growth of bacteria (Atmadja, 1992).
CONCLUSION
Extract Gracilaria sp. have potential as an antibacterial. Based on the content of TVB, TPC and pH, the use of extracts of Gracilaria sp. 2% were able to extend the shelf life of red tilapia fillet during low-temperature storage from day 6 to day 10 of storage.
ACKNOWLEDGMENT
The research was carried out with funding from the Community Fund of the Faculty of Agriculture, Universitas Gadjah Mada Research Grant program through the Faculty of Agriculture Universitas Gadjah Mada 2011, Number: 1556/PN/TU.