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Preparation and use of freshwater fish, rohu (Labeo rohita) protein dispersion in shelf-life extension of the fish steaks
Lebensm.-Wiss. U.-Technol. 36 (2003) 433–439
Preparation and use of freshwater fish, rohu (Labeo rohita) protein dispersion in shelf-life extension of the fish steaks S. Panchavarnama, S. Basua, K. Manishab, S.B. Warrierb,*, V. Venugopalb b
a Central Institute of Fisheries Education, Andheri, Mumbai 400 061, India Food Technology Division, Bhabha Atomic Research Center, Mumbai 400 085, India
Received 16 September 2002; accepted 13 January 2003
Abstract A process to enhance shelf-life of freshwater fish, rohu, on ice using combination treatment of coating the fish steaks with gel dispersion from the same fish and low-dose gamma irradiation is described. Washed pieces of freshwater fish, rohu were converted into gel by dilute acetic acid treatment. The gel was homogenized in water to a dispersion having 29 g/L protein, apparent viscosity of 1.0 Pa s and pH 3.5. Coating of fresh rohu steaks by dipping in the dispersion for 1 h or gamma irradiation at 1 kGy gave a shelflife of 32 days in ice, in comparison to 20 days for the untreated steaks. Irradiation at 1 kGy of the dispersion-coated steaks enhanced their shelf-life to 42 days. Bleaching of the pink colour of the steaks by the treatment was prevented when one of either butylated hydroxy anisole or ascorbic acid was incorporated at 5 g/kg (w/v) in the dispersion. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Freshwater fish; Weak acid-induced gelation; Dispersion; Coating; Gamma irradiation; Shelf-life extension
1. Introduction Gelation is an important functional property of food proteins. Gelation of myosin molecules involves partial denaturation followed by irreversible aggregation of myosin heads through formation of disulphide bonds and helix–coil transition of the tail part of the molecules, resulting in a three-dimensional network (Stone & Stanley, 1992). While most of the studies on gelation of fish proteins have been carried out at near-neutral pH conditions (Stone & Stanley, 1992), information on gelation of these proteins at acidic pH is sparse. We have observed that washed fish muscle structural proteins could form a gel when its pH was lowered to 3.5 by weak organic acids such as acetic or lactic acid. Gelation of shark meat proteins under mild acidic conditions, which could be measured in terms of increase in apparent viscosity, was dependent upon protein concentration and temperature (Venugopal, Doke, & Nair, 1994). The proteins in the gel dispersions in water were stable to heat as well as centrifugation, while presence of salts or increase in pH resulted in precipitation of the *Corresponding author. Fax: +91-22-25505151. E-mail address: [email protected] (S.B. Warrier).
proteins (Venugopal, Doke, & Thomas, 1998). A number of products could be developed such as spraydried protein powders, biodegradable films, etc. making use of the thermostable nature of the proteins in the dispersion (Venugopal, 1997). Fishery products being highly perishable, there is a need for development of processes for their better distribution in ‘as-is’ condition. During the recent years, there has been interest in combination treatment through creation of multiple hurdles to control microbial growth in food products (Leistner & Gorris Leon, 1995; Thakur & Singh, 1994, 1995; Scott, 1989). Edible coatings from polysaccharides, proteins, and also lipids have been recognized to extend quality of fresh, frozen and processed muscle foods including fish by retarding moisture loss, lipid oxidation as well as discolouration, enhancing product appearance in retail packages, and functioning as carriers of food additives (Gennadios, Hanna, & Kurth, 1997; Krochta & Baldwin, 1994). Edible packaging films based on fish myofibrillar proteins has been developed (Venugopal, 1998; Cuq, Aymard, Cuq, & Guilbert, 1995). In spite of numerous studies on various applications, technology for largescale production of edible films remains rather cumbersome, which generally involves drying the ingredient in
0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0023-6438(03)00040-9
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solution in thin layers followed by peeling of the film. Stickiness of protein-based films is another problem in their handling. Direct dipping of the products in the dispersion may alleviate some of these problems. Extensive investigations, worldwide, in the last four decades have shown the beneficial effects of radiation processing for the preservation and hygienization of foods including those from animal origin. Gamma irradiation at low doses is known to extend shelf-life of fresh fish and shellfish by elimination of spoilage causing microorganisms. A dose of 1–3 kGy has been successfully applied to extend refrigerated shelf-life of several fish and shellfish species. (Thakur & Singh, 1994; Venugopal, Doke, & Thomas, 1999). Production of freshwater fish species by aquaculture has increased considerably throughout the world during the recent years. In India, carp species including silver carp, grass carp, common carp, big head, crucial carp, rohu, catla and mrigal constitute more than 80% of freshwater aquaculture production (Ayyappan, 2000). Annual production of rohu (Labeo rohita) is about 60,000 metric tonnes in the country (MPEDA, 2000). Since aquaculture in India is concentrated in certain regions, techniques are required for control of postharvest losses and efficient inland distribution of the produce. In this paper, we report development of aqueous dispersion of rohu meat and its application in combination with low-dose gamma irradiation for extension of chilled shelf-life of the fish steaks.
incubated for 18 h at 0–2 C for gelation of the proteins. The gel was removed from the acetic acid solution and homogenized using a kitchen homogenizer in equal amount of cold water to get the dispersion, having a protein content of 29 g/L and apparent viscosity of 1 Pa s. In aliquots of the dispersion, BHA or ascorbic acid was incorporated at a concentration of 5 g/kg. The prepared dispersion was used for coating of the fish steaks. 2.3. Coating of the steaks with the dispersion A total number of 64 steaks was used for the experiment. For dispersion treatment, 48 steaks were suspended in chilled (0–2 C), dispersion (1.0 kg) in a stainless-steel trough for a period of 1 h, with occasional gentle shaking for uniform coating. After the treatment, the treated steaks were drained and aerobically packaged in sixteen 500 gauge polyethylene pouches, each bag containing two steaks. The packages (8 Nos.) were subjected to gamma irradiation, while the other lot of eight packages served as unirradiated, dispersion treated samples. The third lot 16 dispersion-coated steaks were vacuum packaged in eight pouches and then subjected to gamma irradiation. A fourth lot of 16 steaks in eight aerobic packages served as control, which were subjected to neither dispersion coating nor gamma irradiation. 2.4. BHA and ascorbic acid treatments
2. Materials and methods 2.1. Materials Fresh rohu (L. rohita) were procured from the local market within 24 h of landing and brought to the laboratory in ice. The whole fish were beheaded, eviscerated and cut into steaks of 2.5 cm thickness, each weighing about 42–45 g. Butylated hydroxyanisole (BHA) was obtained from Sigma Chemical Co., St. Louis, MO and l-ascorbic acid, from E. Merck (India) Ltd., Mumbai. Other chemicals used were of analytical grade procured from local manufacturers.
In separate experiments, influence of antioxidants, namely, butyl hydroxyanisole (BHA) or ascorbic acid on colour retention of treated rohu steaks was examined. For this purpose, dispersion containing either BHA or ascorbic acid at a concentration of 5 g/kg was used. BHA was dissolved in traces of alcohol prior to incorporation in the dispersion. Fresh steaks (two numbers each) were treated with the dispersion containing the antioxidant. The samples were either vacuum or air packaged, followed by gamma irradiation. The visual colour of the treated samples was measured, as mentioned below. 2.5. Gamma irradiation
2.2. Preparation of dispersion In order to make dispersion, 12 steaks were cut into pieces of 8–10 g each and the pieces (500 g) were suspended in 1.5 kg cold (o10 C) water and held in a cold room (0–2 C) for 18 h for removal of myoglobin pigments, and other soluble compounds. The washed meat pieces were decanted through a nylon sieve and washed again by rinsing in cold water for 1 h. After washing, the pieces were suspended in cold water containing glacial acetic acid in water at 20 mL/kg and
The packages containing steaks treated with or without dispersion were held under flake ice and irradiated at a dose of 1 kGy using a package irradiator (Atomic Energy of Canada Ltd) with 60Co source, having a dose rate of 0.022 kGy/min. 2.6. Chilled storage The treated samples were stored under ice in a 5 cm thick polystyrene-insulated box (52.2 34 24 cm3). At
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periodic intervals, the packages were removed for quality assessment in terms of biochemical, microbiological and sensory methods. 2.7. Microbiological analyses The fish samples (10 g) from each treatment were aseptically homogenized for 1 min in 90 mL sterile saline in a pre-sterilized Sorvall omnimixer cup. Appropriate serial dilutions of the homogenate were placed in Petri plates in triplicate. The colony forming units (cfu) were determined using plate count agar (Difco, Detroit, MI). The plates were incubated at 30 C for 48 h before counting the colonies. 2.8. Biochemical indices of spoilage Meat portions from two steaks were homogenized and the mince was used for biochemical analyses. An amount of 10 g of the mince was blended with 90 mL of 100 g/L (w/v) aqueous solution of trichloro acetic acid (TCA) in a Sorvall omnimixer cup for 1 min and filtered through Whatmann No. 1 filter paper. The TCA extract was used for determination of total volatile basic nitrogen (TVBN) by Convey microdiffusion method (Farber & Ferro, 1956). The TVBN values were expressed as mg N/kg fish meat. Total volatile acids (TVA) content was determined by steam distillation by the procedure described by Venugopal, Lewis, and Nadkarni (1981). A known portion of the mince was homogenized in presence of phosphotungstic acid and dilute sulphuric acid and the filtrate was steam distilled. TVA number was expressed as the mL of alkali required to neutralize the acids from 1.0 kg of fish meat. Lipid oxidation was determined in terms of 2-thiobarbituric acid (TBA) value, as described by Ghadi and Venugopal (1994). TBA values were expressed as mg malonaldehyde per kg of sample by using a factor of 5.8. 2.9. Visual colour measurement Colour of steaks was measured with a hand-held Minolta Chroma Meter (CR 14 Minolta Chroma Co., Osaka, Japan). A colour scale on Hunter mode (‘L’) was used to measure the degree of whiteness. The instrument was calibrated using a Minolta CR A74 whiteness calibration plate. Six measurements were made per package and the values were expressed as mean7s.d. 2.10. Sensory analysis A panel of six scientists from the Food Technology Division, who were experienced in sensory evaluation of fishery products, evaluated the sensory quality of the samples in terms of odour, during the course of chilled storage. The samples were rinsed once in potable water,
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cooked in a microwave oven for 1 min, brought to ambient temperature (22–23 C) and served to the panel members. A 10-point odour scale (FAO, 1995) was used. The scores were as follows: 10, fresh odour, characteristic of fresh rohu; 9, marginal loss of fresh odour; 8, light loss of fresh odour; 7, definite loss of fresh odour; 6, almost no fresh odour; 5, slight ammonical and rancid odour; 4–2, increasing off odour; and 1, putrid odour. Fish that scored between 10 and 5 were considered acceptable and those scoring less than 5 were spoiled. 2.11. Apparent viscosity measurement The apparent viscosity of the dispersion (200 mL) taken in a 250 mL beaker, held in ice water bath was measured using a Brookfield synchro-lectric viscometer Model RVT (Cooksville, Ontario, Canada) as described by Venugopal et al. (1998). The viscosity was measured using spindle No. 3 at a speed of 50 rpm. The values were recorded after rotation of the spindle for 30 s. The apparent viscosity values were obtained using a conversion factor provided by the manufacturer and were expressed in Pascal seconds (Pa s). The average of two independent experiments were used. 2.12. Proximate composition Protein and moisture of the fish were determined according to AOAC (1990). The protein content was determined by measuring nitrogen by Kjeldahl method using Kjelplus digestion and distillation system (Pelican Instruments, Madras, India) and was expressed as N 6.25. Crude lipid was determined by the procedure of Bligh and Dyer (1959). The values of three independent experiments were recorded as mean7s.d. 2.13. Statistical analysis Statistical analysis of the data was done by the standard methods. Analysis of variance (ANOVA) was employed to find out significance between different treatments and days of storage. Level of significance was determined at 95% ðPo0:05Þ:
3. Results 3.1. Characteristics of the dispersion The fish used had a proximate composition of 779.471/kg moisture, 174.2716/kg protein and 21.974/kg crude lipid. Overnight incubation of the meat pieces in cold water resulted in removal of coloured pigments, and odour-bearing compounds. There was about 50 g/kg increase in weight of the fish during overnight soaking in water. Dipping the washed
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600
control coating 1 kGy coating + 1 kGy
TVBN mg N / kg
500 400 300 200 100 0 0
5
10
15 20 25 30 35 Storage period in ice (days)
40
45
Fig. 1. Influence of protein coating and gamma irradiation on TVBN in rohu steaks during ice storage.
control coating 1 kGy coating+1 kGy
1200 TVA number/kg meat
meat in dilute acetic acid resulted in gelation of the meat proteins, associated with 340 g/kg increase in weight due to hydration of the proteins. While the washed steaks were opaque in appearance, the acetic acid treated steaks were translucent. Presence of NaCl negatively influenced protein hydration. Thus, the weight increase was only 200 g/kg when 2.0 g/kg NaCl was present in the aqueous acetic acid. The imbibed water was strongly held in the protein gel, since no water was separated when the gel was centrifuged at 12,100 g for 10 min. The control and dispersion-coated fish steaks had an initial microbial counts of 5.8 105 and 9.5 105 cfu/g, respectively. The microbial load increased to a value of 1 107 and 5.5 107 cfu/g, during storage for a period of 24 days in the case of both dispersion noncoated and coated steaks, respectively. Irradiation at 1 kGy of steaks coated with or without dispersion decreased the initial load to 1.3 104 and 1.0 104 cfu/g, respectively. After storage for a period of 36 days in ice, microbial counts of these samples reached a value of 1 108 and 5.9 107 cfu/g, respectively. Surface coating of the steaks with the dispersion reduced formation of TVBN during ice storage, as shown in Fig. 1. All the samples showed comparable TVBN values till 17th days of storage. Afterwards, both untreated control and steaks irradiated at 1 kGy showed enhanced TVBN values, indicative of spoilage. However, dispersion-coated steaks showed lower TVBN values as a function of storage period. Irradiation of dispersion-coated steaks further reduced the rate of TVBN formation during chilled storage. Thus, dispersion coating delayed TVBN formation, requiring an ice storage of 36 days to give a TVBN value of 212 mg N/kg, whereas, irradiated dispersion-coated steaks gave a TVBN value of 133 mg N/kg after a period of 42 days (Fig. 1). ANOVA of the TVBN data showed significant ðPo0:05Þ difference between control and irradiation together with dispersion treatment during storage of the steaks.
1000 800 600 400 200 0 0
5
10
15 20 25 30 35 Sorage period in ice (days)
40
45
Fig. 2. Influence of protein coating and gamma irradiation on TVA in rohu steaks during ice storage.
control coating 1 kGy coating+1 kGy
12 mg malonaldehyde/kg
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10 8 6 4 2 0 0
5
10
15 20 25 30 35 Storage period in ice (days)
40
45
Fig. 3. Influence of protein coating and gamma irradiation on TBA value of rohu steaks during ice storage.
Fig. 2 shows the formation of TVA during ice storage. In the case of untreated control, TVA values increased from 4 to 84.8/kg after 24 days of ice storage, while irradiation at 1 kGy resulted suppression of TVA formation, giving a TVA value of 62.7/kg after 42 days of ice storage. The TVA values of dispersion coated alone as well as protein coated and irradiated (1 kGy) samples after 36 days of ice storage were 90.7 and 76, respectively. The initial high TVA values in dispersion treated samples were due to interference of acetic acid, which is present in the dispersion, which is also one of the volatile acids present in spoiled fish (Venugopal et al., 1981). Fig. 3 depicts the extent of lipid oxidation in the steaks in terms of TBA value during ice storage. The TBA values increased to about 12 only after 35 days ice storage in dispersion coated and irradiated samples. The values of other samples were less even after 40 days storage. Fig. 4 depicts the sensory scores of the steaks subjected to coating, irradiation or a combination of both. The odour scores of samples decreased rapidly in the case of untreated steaks during the course of ice
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storage, suggesting rapid spoilage. However, while, dispersion coated or irradiated samples had acceptability for longer period, combination of coating and irradiation gave products maximum shelf-life. Based on odour score, the untreated steaks had a shelf-life of 20 days, irradiation alone or coating gave a shelf-life of 32 days. Combination of dispersion and irradiation gave a shelflife of 42 days. Dispersion treated samples had a sour odour, which could be removed by washing in water. Coated steaks after microwave oven cooking were not sour to taste. There was no irradiation odour also in the products. Statistical analysis of the data showed significant ðPo0:05Þ differences in shelf-life of untreated fish steaks in comparison with dispersion treatment alone or in combination with irradiation. Dispersion treatment either alone or in combination with irradiation, however, had some adverse effect on the colour of the product, as shown in Table 1. While irradiation alone did not bleach the colour, irradiation in combination with dispersion coating resulted in bleaching of the pink colour of the flesh, as observed by higher (>50) Hunter L values. However, bleaching of pigments was prevented by incorporation of antioxidants, namely, BHA or ascorbic acid at 0.5% (w/v) in the dispersion. Alternatively, vacuum packaging also prevented bleaching of the colour.
10
control coating 1 kGy coating+1 kGy
9 Sensory score
8 7 6 5 4 3 2 0
5
10
15
20
25
30
35
40
45
Storage period in ice (days) Fig. 4. Influence of protein coating and gamma irradiation on sensory score of rohu steaks during ice storage.
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4. Discussion The results suggest that a dispersion of washed rohu meat could be prepared through acetic acid treatment of the fish meat. The dispersion could be applied as a surface coating in combination with gamma irradiation at 1 kGy to enhance the shelf-life of the fish steaks on ice. While the effect of low-dose irradiation in shelf-life extension of fishery products including freshwater fish is well documented (Venugopal et al., 1999; Szule, Stamczae, & Peconek, 1990), the present work indicates advantage of coating with a dispersion from the same fish along with radiation. Thus, while irradiation at 1 kGy or dispersion coating alone gave a shelf-life of 32 days for the fish on ice, a combination treatment could enhance the shelf-life up to 42 days. The major preservative in the dispersion is the acetic acid. The protective action of weak organic acids such as lactic as well as acetic acids and also their salts against spoilage causing and pathogenic microorganisms in muscle foods has been documented (Lin & Chuag, 2001; Cuttler & Siragusa, 1994). Shelf stability of cod fillets was found to be enhanced by a dip in carbonic acid and packaging in low oxygen permeable pouches (Daniels & Krishnamurthi, 1986). Similarly, dip in dilute lactic or acetic acid followed by packaging in sterile plastic bag enhanced refrigerated shelf-life of catfish fillets. The treatment suppressed growth of aerobic microorganisms and extended shelf-life up to 16 days. Acetic acid was found to have greater antimicrobial activity than lactic acid (Ingram, 1989; Marshall & Kim, 1996). Similar effect has been reported in the case of Chilean mackerel (Borquez & Peters, 1994). In the present experiments, instead of a dip in acetic acid alone, a protein dispersion was used, since it could provide not only the antimicrobial effect of acetic acid, but also a protective coating of myofibrillar proteins of the same fish on the fish steaks. We have recently observed that coating of frozen mince of mackerel, a fatty fish, with the dispersion prepared from the same fish could control rancidity development and weight loss due to dehydration during frozen storage of the product.
Table 1 Colour of rohu steaks after different treatments and ice storage for 40 days Treatment
Appearance
Hunter value, L
Remark
No treatment Irradiated, 1 kGy Dispersion coating Dispersion+BHA Dispersion+ascorbic acid Dispersion+irradiation Dispersion+irradiation (vacuum pack)
Pinkish Pinkish Bleached Pinkish Pinkish Bleached Pinkish
39.971.9 38.173.6 53.172.2 44.474.0 44.972.3 50.572.1 40.171.6
Putrid odour Putrid odour Sour odour Sour odour Sour odour Sour odour Sour odour
Unless otherwise stated, all the samples were aerobically packaged.
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Fish myofibrillar proteins are known to be soluble only in extractants having high ionic strength at neutral pH. In the present study dissolution of the meat in water was achieved through gelation and associated hydration of the proteins. Fish muscle structural proteins have been shown to undergo gelation in presence of weak organic acids (Venugopal, Kakatkar, Doke, & Bongirwar, 2002a; Chawla, Venugopal, & Nair, 1996). Gelation was accompanied by hydration of the proteins, as also observed by Hermansson (1986). However, gelation and associated hydration required initial soaking the meat in water to wash off low molecular weight components from the structural proteins. This facilitated mild pHinduced conformational changes leading to protein– protein interactions and swelling of the proteins through binding of water in the three-dimensional structure (Venugopal et al., 1994; Stone & Stanley, 1992). The visco-elastic nature of gel formed during mild acidinduced gelation of shark meat has been characterized recently (Venugopal et al., 2002b). Hydration of rohu muscle proteins was inhibited by salt, presumably due to their interference with electrostatic interactions among the proteins (Venugopal, 1997; Hermansson, 1986). Therefore, salt could not be used in the dispersion to enhance the antimicrobial properties of the preparation. Alternatively, low-dose irradiation was used as another hurdle to control microbial growth in the fish (Thakur & Singh, 1994, 1995; Scott, 1989). Surface coating of steaks with the dispersion, however caused some flesh discolouration, presumably by oxidation of the pigments under low pH conditions. Similarly, dipping in dilute acetic acid has also been reported to cause flesh discolouration in catfish fillets (Marshall & Kim, 1996). Lin and Chuang (2001) observed higher Hunter L values indicative of flesh colour bleaching in acetic acid treated pork loin chops. Discolouration of rohu meat was prevented when ascorbic acid or BHA was incorporated in the dispersion. BHA is also known to have antimicrobial activity (Davidson & Branen, 1980). Alternatively, vacuum packaging prevented the discolouration of the fish.
5. Conclusion Washed rohu meat could be converted into a gel by lowering its pH with acetic acid. The gel dispersion could be used as an edible coating to enhance the chilled shelflife of rohu steaks. Irradiation at 1 kGy in conjunction with dispersion coating has additional advantage in extension of shelf-life of the fish steaks on ice.
Acknowledgements The authors thank Dr. S. Ayyappan, Director, Central Institute of Fisheries Education, Mumbai, for
valuable support. The technical assistance of Ms. A. Kakatkar and Ms. Vaishali Mahale is gratefully acknowledged.
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Preparation and use of freshwater fish, rohu (Labeo rohita) protein dispersion in shelf-life extension of the fish steaks S. Panchavarnama, S. Basua, K. Manishab, S.B. Warrierb,*, V. Venugopalb b
a Central Institute of Fisheries Education, Andheri, Mumbai 400 061, India Food Technology Division, Bhabha Atomic Research Center, Mumbai 400 085, India
Received 16 September 2002; accepted 13 January 2003
Abstract A process to enhance shelf-life of freshwater fish, rohu, on ice using combination treatment of coating the fish steaks with gel dispersion from the same fish and low-dose gamma irradiation is described. Washed pieces of freshwater fish, rohu were converted into gel by dilute acetic acid treatment. The gel was homogenized in water to a dispersion having 29 g/L protein, apparent viscosity of 1.0 Pa s and pH 3.5. Coating of fresh rohu steaks by dipping in the dispersion for 1 h or gamma irradiation at 1 kGy gave a shelflife of 32 days in ice, in comparison to 20 days for the untreated steaks. Irradiation at 1 kGy of the dispersion-coated steaks enhanced their shelf-life to 42 days. Bleaching of the pink colour of the steaks by the treatment was prevented when one of either butylated hydroxy anisole or ascorbic acid was incorporated at 5 g/kg (w/v) in the dispersion. r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. Keywords: Freshwater fish; Weak acid-induced gelation; Dispersion; Coating; Gamma irradiation; Shelf-life extension
1. Introduction Gelation is an important functional property of food proteins. Gelation of myosin molecules involves partial denaturation followed by irreversible aggregation of myosin heads through formation of disulphide bonds and helix–coil transition of the tail part of the molecules, resulting in a three-dimensional network (Stone & Stanley, 1992). While most of the studies on gelation of fish proteins have been carried out at near-neutral pH conditions (Stone & Stanley, 1992), information on gelation of these proteins at acidic pH is sparse. We have observed that washed fish muscle structural proteins could form a gel when its pH was lowered to 3.5 by weak organic acids such as acetic or lactic acid. Gelation of shark meat proteins under mild acidic conditions, which could be measured in terms of increase in apparent viscosity, was dependent upon protein concentration and temperature (Venugopal, Doke, & Nair, 1994). The proteins in the gel dispersions in water were stable to heat as well as centrifugation, while presence of salts or increase in pH resulted in precipitation of the *Corresponding author. Fax: +91-22-25505151. E-mail address: [email protected] (S.B. Warrier).
proteins (Venugopal, Doke, & Thomas, 1998). A number of products could be developed such as spraydried protein powders, biodegradable films, etc. making use of the thermostable nature of the proteins in the dispersion (Venugopal, 1997). Fishery products being highly perishable, there is a need for development of processes for their better distribution in ‘as-is’ condition. During the recent years, there has been interest in combination treatment through creation of multiple hurdles to control microbial growth in food products (Leistner & Gorris Leon, 1995; Thakur & Singh, 1994, 1995; Scott, 1989). Edible coatings from polysaccharides, proteins, and also lipids have been recognized to extend quality of fresh, frozen and processed muscle foods including fish by retarding moisture loss, lipid oxidation as well as discolouration, enhancing product appearance in retail packages, and functioning as carriers of food additives (Gennadios, Hanna, & Kurth, 1997; Krochta & Baldwin, 1994). Edible packaging films based on fish myofibrillar proteins has been developed (Venugopal, 1998; Cuq, Aymard, Cuq, & Guilbert, 1995). In spite of numerous studies on various applications, technology for largescale production of edible films remains rather cumbersome, which generally involves drying the ingredient in
0023-6438/03/$30.00 r 2003 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0023-6438(03)00040-9
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solution in thin layers followed by peeling of the film. Stickiness of protein-based films is another problem in their handling. Direct dipping of the products in the dispersion may alleviate some of these problems. Extensive investigations, worldwide, in the last four decades have shown the beneficial effects of radiation processing for the preservation and hygienization of foods including those from animal origin. Gamma irradiation at low doses is known to extend shelf-life of fresh fish and shellfish by elimination of spoilage causing microorganisms. A dose of 1–3 kGy has been successfully applied to extend refrigerated shelf-life of several fish and shellfish species. (Thakur & Singh, 1994; Venugopal, Doke, & Thomas, 1999). Production of freshwater fish species by aquaculture has increased considerably throughout the world during the recent years. In India, carp species including silver carp, grass carp, common carp, big head, crucial carp, rohu, catla and mrigal constitute more than 80% of freshwater aquaculture production (Ayyappan, 2000). Annual production of rohu (Labeo rohita) is about 60,000 metric tonnes in the country (MPEDA, 2000). Since aquaculture in India is concentrated in certain regions, techniques are required for control of postharvest losses and efficient inland distribution of the produce. In this paper, we report development of aqueous dispersion of rohu meat and its application in combination with low-dose gamma irradiation for extension of chilled shelf-life of the fish steaks.
incubated for 18 h at 0–2 C for gelation of the proteins. The gel was removed from the acetic acid solution and homogenized using a kitchen homogenizer in equal amount of cold water to get the dispersion, having a protein content of 29 g/L and apparent viscosity of 1 Pa s. In aliquots of the dispersion, BHA or ascorbic acid was incorporated at a concentration of 5 g/kg. The prepared dispersion was used for coating of the fish steaks. 2.3. Coating of the steaks with the dispersion A total number of 64 steaks was used for the experiment. For dispersion treatment, 48 steaks were suspended in chilled (0–2 C), dispersion (1.0 kg) in a stainless-steel trough for a period of 1 h, with occasional gentle shaking for uniform coating. After the treatment, the treated steaks were drained and aerobically packaged in sixteen 500 gauge polyethylene pouches, each bag containing two steaks. The packages (8 Nos.) were subjected to gamma irradiation, while the other lot of eight packages served as unirradiated, dispersion treated samples. The third lot 16 dispersion-coated steaks were vacuum packaged in eight pouches and then subjected to gamma irradiation. A fourth lot of 16 steaks in eight aerobic packages served as control, which were subjected to neither dispersion coating nor gamma irradiation. 2.4. BHA and ascorbic acid treatments
2. Materials and methods 2.1. Materials Fresh rohu (L. rohita) were procured from the local market within 24 h of landing and brought to the laboratory in ice. The whole fish were beheaded, eviscerated and cut into steaks of 2.5 cm thickness, each weighing about 42–45 g. Butylated hydroxyanisole (BHA) was obtained from Sigma Chemical Co., St. Louis, MO and l-ascorbic acid, from E. Merck (India) Ltd., Mumbai. Other chemicals used were of analytical grade procured from local manufacturers.
In separate experiments, influence of antioxidants, namely, butyl hydroxyanisole (BHA) or ascorbic acid on colour retention of treated rohu steaks was examined. For this purpose, dispersion containing either BHA or ascorbic acid at a concentration of 5 g/kg was used. BHA was dissolved in traces of alcohol prior to incorporation in the dispersion. Fresh steaks (two numbers each) were treated with the dispersion containing the antioxidant. The samples were either vacuum or air packaged, followed by gamma irradiation. The visual colour of the treated samples was measured, as mentioned below. 2.5. Gamma irradiation
2.2. Preparation of dispersion In order to make dispersion, 12 steaks were cut into pieces of 8–10 g each and the pieces (500 g) were suspended in 1.5 kg cold (o10 C) water and held in a cold room (0–2 C) for 18 h for removal of myoglobin pigments, and other soluble compounds. The washed meat pieces were decanted through a nylon sieve and washed again by rinsing in cold water for 1 h. After washing, the pieces were suspended in cold water containing glacial acetic acid in water at 20 mL/kg and
The packages containing steaks treated with or without dispersion were held under flake ice and irradiated at a dose of 1 kGy using a package irradiator (Atomic Energy of Canada Ltd) with 60Co source, having a dose rate of 0.022 kGy/min. 2.6. Chilled storage The treated samples were stored under ice in a 5 cm thick polystyrene-insulated box (52.2 34 24 cm3). At
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periodic intervals, the packages were removed for quality assessment in terms of biochemical, microbiological and sensory methods. 2.7. Microbiological analyses The fish samples (10 g) from each treatment were aseptically homogenized for 1 min in 90 mL sterile saline in a pre-sterilized Sorvall omnimixer cup. Appropriate serial dilutions of the homogenate were placed in Petri plates in triplicate. The colony forming units (cfu) were determined using plate count agar (Difco, Detroit, MI). The plates were incubated at 30 C for 48 h before counting the colonies. 2.8. Biochemical indices of spoilage Meat portions from two steaks were homogenized and the mince was used for biochemical analyses. An amount of 10 g of the mince was blended with 90 mL of 100 g/L (w/v) aqueous solution of trichloro acetic acid (TCA) in a Sorvall omnimixer cup for 1 min and filtered through Whatmann No. 1 filter paper. The TCA extract was used for determination of total volatile basic nitrogen (TVBN) by Convey microdiffusion method (Farber & Ferro, 1956). The TVBN values were expressed as mg N/kg fish meat. Total volatile acids (TVA) content was determined by steam distillation by the procedure described by Venugopal, Lewis, and Nadkarni (1981). A known portion of the mince was homogenized in presence of phosphotungstic acid and dilute sulphuric acid and the filtrate was steam distilled. TVA number was expressed as the mL of alkali required to neutralize the acids from 1.0 kg of fish meat. Lipid oxidation was determined in terms of 2-thiobarbituric acid (TBA) value, as described by Ghadi and Venugopal (1994). TBA values were expressed as mg malonaldehyde per kg of sample by using a factor of 5.8. 2.9. Visual colour measurement Colour of steaks was measured with a hand-held Minolta Chroma Meter (CR 14 Minolta Chroma Co., Osaka, Japan). A colour scale on Hunter mode (‘L’) was used to measure the degree of whiteness. The instrument was calibrated using a Minolta CR A74 whiteness calibration plate. Six measurements were made per package and the values were expressed as mean7s.d. 2.10. Sensory analysis A panel of six scientists from the Food Technology Division, who were experienced in sensory evaluation of fishery products, evaluated the sensory quality of the samples in terms of odour, during the course of chilled storage. The samples were rinsed once in potable water,
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cooked in a microwave oven for 1 min, brought to ambient temperature (22–23 C) and served to the panel members. A 10-point odour scale (FAO, 1995) was used. The scores were as follows: 10, fresh odour, characteristic of fresh rohu; 9, marginal loss of fresh odour; 8, light loss of fresh odour; 7, definite loss of fresh odour; 6, almost no fresh odour; 5, slight ammonical and rancid odour; 4–2, increasing off odour; and 1, putrid odour. Fish that scored between 10 and 5 were considered acceptable and those scoring less than 5 were spoiled. 2.11. Apparent viscosity measurement The apparent viscosity of the dispersion (200 mL) taken in a 250 mL beaker, held in ice water bath was measured using a Brookfield synchro-lectric viscometer Model RVT (Cooksville, Ontario, Canada) as described by Venugopal et al. (1998). The viscosity was measured using spindle No. 3 at a speed of 50 rpm. The values were recorded after rotation of the spindle for 30 s. The apparent viscosity values were obtained using a conversion factor provided by the manufacturer and were expressed in Pascal seconds (Pa s). The average of two independent experiments were used. 2.12. Proximate composition Protein and moisture of the fish were determined according to AOAC (1990). The protein content was determined by measuring nitrogen by Kjeldahl method using Kjelplus digestion and distillation system (Pelican Instruments, Madras, India) and was expressed as N 6.25. Crude lipid was determined by the procedure of Bligh and Dyer (1959). The values of three independent experiments were recorded as mean7s.d. 2.13. Statistical analysis Statistical analysis of the data was done by the standard methods. Analysis of variance (ANOVA) was employed to find out significance between different treatments and days of storage. Level of significance was determined at 95% ðPo0:05Þ:
3. Results 3.1. Characteristics of the dispersion The fish used had a proximate composition of 779.471/kg moisture, 174.2716/kg protein and 21.974/kg crude lipid. Overnight incubation of the meat pieces in cold water resulted in removal of coloured pigments, and odour-bearing compounds. There was about 50 g/kg increase in weight of the fish during overnight soaking in water. Dipping the washed
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600
control coating 1 kGy coating + 1 kGy
TVBN mg N / kg
500 400 300 200 100 0 0
5
10
15 20 25 30 35 Storage period in ice (days)
40
45
Fig. 1. Influence of protein coating and gamma irradiation on TVBN in rohu steaks during ice storage.
control coating 1 kGy coating+1 kGy
1200 TVA number/kg meat
meat in dilute acetic acid resulted in gelation of the meat proteins, associated with 340 g/kg increase in weight due to hydration of the proteins. While the washed steaks were opaque in appearance, the acetic acid treated steaks were translucent. Presence of NaCl negatively influenced protein hydration. Thus, the weight increase was only 200 g/kg when 2.0 g/kg NaCl was present in the aqueous acetic acid. The imbibed water was strongly held in the protein gel, since no water was separated when the gel was centrifuged at 12,100 g for 10 min. The control and dispersion-coated fish steaks had an initial microbial counts of 5.8 105 and 9.5 105 cfu/g, respectively. The microbial load increased to a value of 1 107 and 5.5 107 cfu/g, during storage for a period of 24 days in the case of both dispersion noncoated and coated steaks, respectively. Irradiation at 1 kGy of steaks coated with or without dispersion decreased the initial load to 1.3 104 and 1.0 104 cfu/g, respectively. After storage for a period of 36 days in ice, microbial counts of these samples reached a value of 1 108 and 5.9 107 cfu/g, respectively. Surface coating of the steaks with the dispersion reduced formation of TVBN during ice storage, as shown in Fig. 1. All the samples showed comparable TVBN values till 17th days of storage. Afterwards, both untreated control and steaks irradiated at 1 kGy showed enhanced TVBN values, indicative of spoilage. However, dispersion-coated steaks showed lower TVBN values as a function of storage period. Irradiation of dispersion-coated steaks further reduced the rate of TVBN formation during chilled storage. Thus, dispersion coating delayed TVBN formation, requiring an ice storage of 36 days to give a TVBN value of 212 mg N/kg, whereas, irradiated dispersion-coated steaks gave a TVBN value of 133 mg N/kg after a period of 42 days (Fig. 1). ANOVA of the TVBN data showed significant ðPo0:05Þ difference between control and irradiation together with dispersion treatment during storage of the steaks.
1000 800 600 400 200 0 0
5
10
15 20 25 30 35 Sorage period in ice (days)
40
45
Fig. 2. Influence of protein coating and gamma irradiation on TVA in rohu steaks during ice storage.
control coating 1 kGy coating+1 kGy
12 mg malonaldehyde/kg
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10 8 6 4 2 0 0
5
10
15 20 25 30 35 Storage period in ice (days)
40
45
Fig. 3. Influence of protein coating and gamma irradiation on TBA value of rohu steaks during ice storage.
Fig. 2 shows the formation of TVA during ice storage. In the case of untreated control, TVA values increased from 4 to 84.8/kg after 24 days of ice storage, while irradiation at 1 kGy resulted suppression of TVA formation, giving a TVA value of 62.7/kg after 42 days of ice storage. The TVA values of dispersion coated alone as well as protein coated and irradiated (1 kGy) samples after 36 days of ice storage were 90.7 and 76, respectively. The initial high TVA values in dispersion treated samples were due to interference of acetic acid, which is present in the dispersion, which is also one of the volatile acids present in spoiled fish (Venugopal et al., 1981). Fig. 3 depicts the extent of lipid oxidation in the steaks in terms of TBA value during ice storage. The TBA values increased to about 12 only after 35 days ice storage in dispersion coated and irradiated samples. The values of other samples were less even after 40 days storage. Fig. 4 depicts the sensory scores of the steaks subjected to coating, irradiation or a combination of both. The odour scores of samples decreased rapidly in the case of untreated steaks during the course of ice
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storage, suggesting rapid spoilage. However, while, dispersion coated or irradiated samples had acceptability for longer period, combination of coating and irradiation gave products maximum shelf-life. Based on odour score, the untreated steaks had a shelf-life of 20 days, irradiation alone or coating gave a shelf-life of 32 days. Combination of dispersion and irradiation gave a shelflife of 42 days. Dispersion treated samples had a sour odour, which could be removed by washing in water. Coated steaks after microwave oven cooking were not sour to taste. There was no irradiation odour also in the products. Statistical analysis of the data showed significant ðPo0:05Þ differences in shelf-life of untreated fish steaks in comparison with dispersion treatment alone or in combination with irradiation. Dispersion treatment either alone or in combination with irradiation, however, had some adverse effect on the colour of the product, as shown in Table 1. While irradiation alone did not bleach the colour, irradiation in combination with dispersion coating resulted in bleaching of the pink colour of the flesh, as observed by higher (>50) Hunter L values. However, bleaching of pigments was prevented by incorporation of antioxidants, namely, BHA or ascorbic acid at 0.5% (w/v) in the dispersion. Alternatively, vacuum packaging also prevented bleaching of the colour.
10
control coating 1 kGy coating+1 kGy
9 Sensory score
8 7 6 5 4 3 2 0
5
10
15
20
25
30
35
40
45
Storage period in ice (days) Fig. 4. Influence of protein coating and gamma irradiation on sensory score of rohu steaks during ice storage.
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4. Discussion The results suggest that a dispersion of washed rohu meat could be prepared through acetic acid treatment of the fish meat. The dispersion could be applied as a surface coating in combination with gamma irradiation at 1 kGy to enhance the shelf-life of the fish steaks on ice. While the effect of low-dose irradiation in shelf-life extension of fishery products including freshwater fish is well documented (Venugopal et al., 1999; Szule, Stamczae, & Peconek, 1990), the present work indicates advantage of coating with a dispersion from the same fish along with radiation. Thus, while irradiation at 1 kGy or dispersion coating alone gave a shelf-life of 32 days for the fish on ice, a combination treatment could enhance the shelf-life up to 42 days. The major preservative in the dispersion is the acetic acid. The protective action of weak organic acids such as lactic as well as acetic acids and also their salts against spoilage causing and pathogenic microorganisms in muscle foods has been documented (Lin & Chuag, 2001; Cuttler & Siragusa, 1994). Shelf stability of cod fillets was found to be enhanced by a dip in carbonic acid and packaging in low oxygen permeable pouches (Daniels & Krishnamurthi, 1986). Similarly, dip in dilute lactic or acetic acid followed by packaging in sterile plastic bag enhanced refrigerated shelf-life of catfish fillets. The treatment suppressed growth of aerobic microorganisms and extended shelf-life up to 16 days. Acetic acid was found to have greater antimicrobial activity than lactic acid (Ingram, 1989; Marshall & Kim, 1996). Similar effect has been reported in the case of Chilean mackerel (Borquez & Peters, 1994). In the present experiments, instead of a dip in acetic acid alone, a protein dispersion was used, since it could provide not only the antimicrobial effect of acetic acid, but also a protective coating of myofibrillar proteins of the same fish on the fish steaks. We have recently observed that coating of frozen mince of mackerel, a fatty fish, with the dispersion prepared from the same fish could control rancidity development and weight loss due to dehydration during frozen storage of the product.
Table 1 Colour of rohu steaks after different treatments and ice storage for 40 days Treatment
Appearance
Hunter value, L
Remark
No treatment Irradiated, 1 kGy Dispersion coating Dispersion+BHA Dispersion+ascorbic acid Dispersion+irradiation Dispersion+irradiation (vacuum pack)
Pinkish Pinkish Bleached Pinkish Pinkish Bleached Pinkish
39.971.9 38.173.6 53.172.2 44.474.0 44.972.3 50.572.1 40.171.6
Putrid odour Putrid odour Sour odour Sour odour Sour odour Sour odour Sour odour
Unless otherwise stated, all the samples were aerobically packaged.
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Fish myofibrillar proteins are known to be soluble only in extractants having high ionic strength at neutral pH. In the present study dissolution of the meat in water was achieved through gelation and associated hydration of the proteins. Fish muscle structural proteins have been shown to undergo gelation in presence of weak organic acids (Venugopal, Kakatkar, Doke, & Bongirwar, 2002a; Chawla, Venugopal, & Nair, 1996). Gelation was accompanied by hydration of the proteins, as also observed by Hermansson (1986). However, gelation and associated hydration required initial soaking the meat in water to wash off low molecular weight components from the structural proteins. This facilitated mild pHinduced conformational changes leading to protein– protein interactions and swelling of the proteins through binding of water in the three-dimensional structure (Venugopal et al., 1994; Stone & Stanley, 1992). The visco-elastic nature of gel formed during mild acidinduced gelation of shark meat has been characterized recently (Venugopal et al., 2002b). Hydration of rohu muscle proteins was inhibited by salt, presumably due to their interference with electrostatic interactions among the proteins (Venugopal, 1997; Hermansson, 1986). Therefore, salt could not be used in the dispersion to enhance the antimicrobial properties of the preparation. Alternatively, low-dose irradiation was used as another hurdle to control microbial growth in the fish (Thakur & Singh, 1994, 1995; Scott, 1989). Surface coating of steaks with the dispersion, however caused some flesh discolouration, presumably by oxidation of the pigments under low pH conditions. Similarly, dipping in dilute acetic acid has also been reported to cause flesh discolouration in catfish fillets (Marshall & Kim, 1996). Lin and Chuang (2001) observed higher Hunter L values indicative of flesh colour bleaching in acetic acid treated pork loin chops. Discolouration of rohu meat was prevented when ascorbic acid or BHA was incorporated in the dispersion. BHA is also known to have antimicrobial activity (Davidson & Branen, 1980). Alternatively, vacuum packaging prevented the discolouration of the fish.
5. Conclusion Washed rohu meat could be converted into a gel by lowering its pH with acetic acid. The gel dispersion could be used as an edible coating to enhance the chilled shelflife of rohu steaks. Irradiation at 1 kGy in conjunction with dispersion coating has additional advantage in extension of shelf-life of the fish steaks on ice.
Acknowledgements The authors thank Dr. S. Ayyappan, Director, Central Institute of Fisheries Education, Mumbai, for
valuable support. The technical assistance of Ms. A. Kakatkar and Ms. Vaishali Mahale is gratefully acknowledged.
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