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Food and Bioproducts Processing

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Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus)
Jayappa M. Koli a , Subrata Basu a , Binay B. Nayak a , Surendra B. Patange b , Ashif U. Pagarkar b , Venkateshwarlu Gudipati a,
a b

Central Institute of Fisheries Education, Versova, Mumbai 400061, India College of Fisheries, Shirgaon, Ratnagiri, Maharashtra, India

a b s t r a c t
The utilization of waste from sh processing industry for production of value added products has attracted substantial attention. Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus) are used for surimi production and have the potential of abundant supply of raw skins and bones. In order to evaluate the waste from Tiger-toothed croaker and Pink perch as a source of gelatin, the gelatin was extracted from skin and bones and its rheological and functional properties were examined. The skins of Tiger-toothed croaker and Pink perch yielded 7.56% and 5.57% gelatin, whereas their bones yielded 4.57% and 3.55% respectively indicating skin as an important source for gelatin production. The gel strength of gelatins from the skins and bones of Tiger-toothed croaker (170 g and 140 g respectively) were found higher than Pink perch skin and bone gelatins (150 g and 130 g respectively). Similarly, the viscosity, melting point, emulsifying capacity and stability, foaming capacity and stability, and water holding capacity of gelatin extracted from Tiger-toothed croaker were in general greater than those of the gelatin from Pink perch and the values of skin gelatin were higher compared to bone gelatin in both the species. Hydroxyproline contents in skin and bone of Tiger-toothed croaker were 7.77 and 7.51 mg/g and in Pink perch they were 7.63 and 7.41 mg/g respectively. It can be concluded from the present study that Tiger-toothed croaker skin is a prospective source to produce gelatin in good yield with desirable functional properties comparable to commercially available mammalian gelatins. 2011 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Gelatin; Tiger-toothed croaker; Pink perch; Gel strength; Viscosity and melting point

1.

Introduction

Gelatin is a heterogenous mixture of high molecular weight water soluble proteins derived from collagen by heat denaturation. It is extensively used as an ingredient to increase the viscosity of aqueous system and form aqueous gels. Traditional sources of gelatin are mainly pig skin and cow hide. In the food industry, gelatin is utilized in confections mainly for providing chewiness, texture, and foam stabilization; in low-fat spreads to provide creaminess, fat reduction, and mouth feel; in dairy to provide stabilization and texturization; in baked goods to provide emulsication, gelling, and stabilization and meat products to provide

water-binding (Johnston-Banks, 1990; Schrieber and Gareis, 2007). Gelatin is normally recommended to enhance protein levels in foodstuffs, and especially in body-building foods. In addition, gelatin is also used to reduce carbohydrate levels in foods formulated for diabetic patients (Gans, 2007). In the pharmaceutical industry, gelatin is used as a matrix for implants, in injectable drug delivery micro spheres, and in intravenous infusions (Pollack, 1990; Rao, 1995; Saddler and Horsey, 1987). There are also reports in which live attenuated viral vaccines used for immunization against measles, mumps, rubella, Japanese encephalitis, rabies, diphtheria, and tetanus contain gelatin as a stabilizer (Burke et al., 1999).

Corresponding author. Tel.: +91 9869719313; fax: +91 2226361573. E-mail address: venkaticar@rediffmail.com (V. Gudipati). Received 25 July 2010; Received in revised form 16 July 2011; Accepted 3 August 2011 0960-3085/$ see front matter 2011 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.fbp.2011.08.001
Please cite this article in press as: Koli, J.M., et al., Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus). Food Bioprod Process (2011), doi:10.1016/j.fbp.2011.08.001

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Gelatin is also widely used for the manufacture of hard and soft capsules, plasma expanders, and in wound care. The global demand for gelatin has been increasing over the years. Recent reports indicate that the annual world output of gelatin is nearly 326,000 tons, with pig skin-derived gelatin accounting for the highest (46%) of output, followed by bovine hides (29.4%), bones (23.1%), and other sources (1.5%) (GME, 2005). Although gelatin has such a wide range of useful applications, strong concerns still persist among consumers with regard to its usage (Asher, 1999). This is mainly due to religious sentiments (both Judaism and Islam forbid the consumption of any pork-related products, while Hindus do not consume cow-related products) as well as the enhanced adherence to vegetarianism throughout the world. In addition, there is an increasing concern among researchers about whether animal tissue-derived collagens and gelatins are capable of transmitting pathogenic vectors such as prions (Wilesmith et al., 1991). However, studies conducted by various authorities have shown that the production process of gelatin is an effective barrier against possible Bovine Spongiform Encephalopathy (BSE) prions (Schrieber and Gareis, 2007). Although porcine gelatin accounts for the highest levels of production, a significant amount of gelatin used in the food and pharmaceutical industries is also derived from cows. The BSE episode, as well as religious concerns, has led to intensive research to identify and develop alternatives to mammal-derived gelatin. Furthermore, strong competition exists among manufacturers for the procurement of pig skin or other mammalian sources, which has created increased demand and raised costs. Researchers are not only continually searching for an alternative to gelatin, and also to nd new sources of gelatin. Within the past decade, there has been increased interest in the market in gelatin derived from sh and poultry. Poultry skin and bones are expected to yield gelatin in the near future, but commercial production is currently limited by low yields (Schrieber and Gareis, 2007). In this background, sh gelatin has been highlighted as a better alternative to mammalian gelatins, from ethical and religious point of views. However, with qualities such as a lower melting point, resulting in faster dissolution in the mouth and absence of residual chewy mouthfeel has affected its commercial applications. Therefore, the production of sh gelatin is still in its infancy, contributing only about 1% of the annual world gelatin production (Arnesen and Gildberg, 2006). Fish used as human food accounts for 78% of the total sh catch, leaving about 21% for non-food uses (Vannuccini, 2004). Processing leads to the generation of a large biomass of sh waste (e.g., skin, bones, and ns), which is generally discarded (7.3 million tons/year) (Kelleher, 2005). Consequently, research has been initiated to investigate an increased utilization of collagenous sh waste for the production of gelatin (Gilsenan and Ross-Murphy, 1999; Holzer, 1996; Nagai and Suzuki, 2000; Wasswa et al., 2007). Fish gelatin is acceptable for Islam, and can be used with minimal restrictions in Judaism and Hinduism. Furthermore, sh skin, which is a major byproduct of the sh-processing industry, could provide a valuable source of gelatin (Badii and Howell, 2006). Morrissey et al. (2005) reported that the solid waste from surimi processing constituted up to 5070% of the original raw material, depending on the method of meat extraction from the carcass. Gudmundsson and Hafsteinsson (1997) and Choi and Regenstein (2000) suggested that in addition to producing sh gelatins to meet religious needs, the commercial use of sh skin and bones, which are normally

discarded, is a good waste management practice leading to additional economic benet. Therefore, the aims of this work were to determine the potential of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus) waste (skin and bone) for production of gelatin and evaluating their rheological and functional properties.

2.
2.1.

Materials and methods

Materials

Freshly generated sh waste (skin and bones) of Tiger-toothed croaker and Pink perch were obtained from Ulka seafood located at Taloja, Panvel, Maharashtra (India). The samples were stored at 20 C until further use. All chemicals used were of analytical grade and purchased from local suppliers.

2.2.

Proximate composition

Proximate composition of raw materials and extracted gelatin were analyzed by measuring moisture, ash, protein and fat contents according to AOAC ofcial methods (AOAC, 1995). The pH value of raw sh skin was measured using the British Standard Institution method, BSI 757 (1975). Fish skins were chopped and blended in distilled water to form 1% (w/v) skin suspension.

2.3.

Gelatin extraction

Gelatin was extracted following the procedure described by Gudmundsson and Hafsteinsson (1997) with some modications. Thawed skin and bones were thoroughly cleaned with excess water to remove superuous material. The cleaned materials were then sequentially soaked with 0.2% (w/v) sodium hydroxide, 0.2% (w/v) sulphuric acid and 1.0% (w/v) citric acid for 40 min. After each soaking treatment, the skins and bones were washed under running tap water until they had a pH of about 7 before transferring to new solution. This cycle was repeated three times with a total time of 2 h for each set of treatment. The ratio of skins and bones to washing liquid used was 1 kg skins and bones (wet weight) to 7 L of acid or alkali solution for each treatment. The skins and bones were then subjected to a nal wash with distilled water to remove any residual matter. The nal extraction was carried out in 3 volumes of distilled water at 45 C for 12 h. The clear extract obtained was ltered with Whatman lter paper (no. 1), using a Buchner funnel. The ltrate was then kept in a tray and dried in oven at 60 C for 16 h. The thin lm of dried matter was powdered, weighed and packed in Zip pack bags, stored at ambient temperature (25 2 C) for further study. The yield of gelatin was calculated on wet weight basis of raw material and expressed as percentage yield.

2.4.

Hydroxyproline content

Hydroxyproline content of gelatin was determined according to the method of Bergman and Loxley (1963) with a slight modication. The samples were hydrolyzed with 6 M HCl at 110 C for 24 h in reex condenser and ltered through Whatman no. 1 lter paper. The ltrate was neutralized with 1 M NaOH to pH 6.06.5. The neutralized sample (0.1 ml) was transferred into a test tube and isopropanol (0.2 ml) was added and mixed well. To the mixture, 0.1 ml of an oxidant solution (a mixture of 7% (w/v) chloroamine T and acetate/citrate buffer, pH 6, at

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a ratio of 1:4 (v/v)) was added and mixed thoroughly. Then 1.3 ml of Ehrlichs reagent solution (a mixture of solution 2 g of p-dimethylamino benzaldehyde in 3 ml of 60% (v/v) perchloric acid (w/v)) and 13 ml of isopropanol) were added. The mixture was mixed and heated at 60 C for 25 min in water bath and then cooled for 23 min in running water. The solution was diluted to 5 ml with isopropanol. Absorbance was measured against water at 558 nm using a spectrophotometer (Thermo spectronic, UV 100628). Hydroxyproline standard solutions, with concentrations ranging from 10 to 60 ppm, were also run simultaneously. Hydroxyproline content was calculated and expressed as mg/g sample.

and 50 ml of sunower oil. The gelatin samples were dispersed with a homogenizer/blender. Each blended sample was divided equally into 50 ml centrifuge tubes. One centrifuge tube was directly centrifuged at 4000 g for 10 min while the other was centrifuged under the same conditions after heating in a water bath at 80 C for 30 min and cooling to room temperature (25 C). The height of the emulsied layer, as a percentage of the total height of material in the unheated tubes, was used to calculate the emulsifying activity and stability, using the following formulae: Emulsion capacity (%) = Height of emulsion layer 100 Height of whole layer

2.5.

Determination of gel strength

Emulsion stability (%) =

Height of emulsion layer after heating 100 Height of whole layer

The gelatin gel was prepared and the bloom value (gel strength) of gelatin gel was determined according to the method described by Wainewright (1977). The gel was prepared in bloom jar (150 ml capacity) by dissolving a 6.67% (w/v) dry gelatin powder in distilled water at 60 C. Then it was cooled for 15 min at room temperature and kept at 7 C for 18 h for maturation. Gel strength was determined on TA.XT2i Texture Analyzer (Stable Micro Systems, Surrey, U.K.) according to British Standard BS 757 (BSI, 1975), with a load cell of 5 kg, cross-head speed 1 mm/s and using a 0.5 in. diameter, at bottomed plunger. The standard glass bloom jar was placed centrally under the plunger and the penetration test was then performed. The maximum force (g) was determined till the probe penetrated into the gel to a depth of 4 mm.

2.9.

Foaming properties

The method of Miller and Goninger (1976) was used to determine foaming properties. The gelatin powder (1 g) was added to 100 ml of distilled water and homogenized for 1 min at high speed (Homogenizer - Polytron - PT - MR2100, Switzerland). The mixture was carefully transferred into a 250 ml calibrated beaker for volume measurement. The foam was calculated as the volume of mixture after blending compared to the original volume. The foaming stability was the ratio of the foam capacity after 30 min divided by the original foam capacity.

2.10. 2.6. Determination of melting point of gelatin

Water holding capacity

The melting point measurement was done by a method modied from Wainewright (1977). Gelatin solutions, 6.67% (w/w), were prepared and a 5 ml aliquot of each sample was transferred to a small glass tube (borosilicate tube, 12 mm 75 mm). The samples were degassed in vacuum desiccators for 5 min. The tubes were then covered with Para lm and heated in a water bath at 60 C for 15 min. The tubes were immediately cooled in ice-chilled water and matured at 10 C for 18 h. Five drops of a mixture of 75% chloroform and 25% reddish brown dye (food colour) was placed on the surface of the gel. The gels were put in a water bath at 10 C and the bath was heated at the rate of 0.20.4 C/min. The temperature of the bath was read using an electronic digital thermometer (Fisher Scientic). The temperature at which the dye drops began to move freely down the gel was taken as the melting point.

Water holding capacity (WHC) was determined using the centrifugation method (Diniz and Martin, 1997). Duplicate samples (0.5 g) of gelatin were dissolved in 20 ml of water in centrifuge tubes and dispersed with a vortex mixer for 30 s. The dispersion was allowed to stand at room temperature for 6 h, and then centrifuged at 2800 g for 30 min. The supernatant was ltered with Whatman No. 1 lter paper and the volume recovered was measured. The difference between the initial volume of distilled water added to the protein sample and the volume of the supernatant was determined, and the results were reported as ml of water absorbed per gram of gelatin sample.

2.11.

Determination of gelatin colour and gel clarity

2.7.

Determination of viscosity

Viscosity of gelatin sample was determined according to the method of Cho et al. (2005). Gelatin solutions (10 g/100 ml) were prepared by dissolving the dry powder in distilled water and heating at 60 C. Viscosity was determined using a Brookeld digital viscometer (Model LV-DV-II, Brookeld Engineering; MA, USA) equipped with No.1 spindle (Model LV) at 60 rpm at 40 1 C. The viscosity was read and reported in terms of cP.

Colour measurement was made by using a Hunter LabScan XE colorimeter (Hunter Associates Laboratory, Inc., VA, USA). The tristimulus L*a*b* measurement mode was used as it relates to the human eye response to colour. The L* variable represents lightness (L* = 0 for black, L* = 100 for white), the a* scale represents the red/green (+a* intensity in red and a* intensity in green) and the b* scale represents the yellow/blue (+b* intensity in yellow and b* intensity in blue). The samples were lled into clear Petri dish and readings were then taken. Clarity was determined by measuring transmittance (%T) at 620 nm in spectrophotometer (Thermo spectronic, Cambridge, U.K) through 6.67% (w/w) gelatin solutions which were heated at 60 C for 1 h (Avena-Bustillos et al., 2006).

2.8.

Emulsifying capacity and stability 2.12. Statistical analysis

The method of Yasumatsu et al. (1972) was used to determine emulsifying capacity and stability. Emulsions were prepared with 1 g of each sample, 50 ml of cold distilled water (4 C)

All data were analyzed for the analysis of variance (ANOVA) and Duncans Multiple Test was carried out to determine the

Please cite this article in press as: Koli, J.M., et al., Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus). Food Bioprod Process (2011), doi:10.1016/j.fbp.2011.08.001

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signicance of difference between the means. Statistical package used in the study was SPSS version 16.0. The difference of means between pairs was resolved by means of condence intervals using post hoc test at level of signicance of p < 0.05. All data represented are the means of triplicates.

swelling. A high degree of cross linking via covalent bonds can cause decrease in solubility of collagen and leading to lower content of extractable gelatin (Foegeding et al., 1996).

3.3.

pH of extracted gelatins

3.
3.1.

Results and discussion

Proximate composition

The proximate compositions of raw materials and extracted gelatin were given in Tables 1 and 2. The gelatins extracted from skins of both the species showed high values of proteins and low values for moisture and fat content. Tiger toothed croaker gelatin contained higher content of protein than Pink perch gelatins i.e. 86.45%, 72.63% for skins and 82.50%, 69.49% for bones respectively. Jongjareonrak et al. (2006) reported a protein content of 87.9% and 88.6% in gelatin extracted from the skin of big eye snapper and brown eye snapper respectively. The gelatin from the skin of adult Nile perch also contained 88% protein when extracted at 50 C (Muyonga et al., 2004). Moisture content of gelatin extracted from Tiger toothed croaker and Pink perch skins were 8.73% and 9.6% respectively, while for croaker and perch bones were 9.5% and 10.33% respectively. Moisture content of all the samples was well below the limit prescribed for edible gelatin i.e. 15% (GME, 2005). At 13% moisture, the glass transition temperature of gelatin is about 64 C which allows particle size reduction to be simple operation (McCormick, 1987). In addition, at 13% moisture content and 25 C gelatin is close to equilibrium with ambient moisture contents of 46% RH. At 68% moisture, gelatin is very hygroscopic and it becomes difcult to determine the physicochemical attributes with accuracy (Cole, 2000). The ash content in the croaker and perch skin gelatins were in the range of 0.301.88% and these values are much less than the recommended maximum limit of 2.6% (Jones, 1977) and the limit given for edible gelatin (2%) (GME, 2005). However, ash content in Tiger-toothed croaker and Pink perch bones were in the range of 2.702.80%, which were higher than the recommended maximum limits indicating non viability of gelatin from bones for utilization as food ingredient.

The pH of extracted gelatins varied between 4.62 and 4.80 (Table 3) indicating their category as Type B. It has been reported that alkali pretreatment results in Type B gelatin with pH in the range of 4 to 5 (Baziwane and He, 2003) and in the present study an alkali pretreatment was employed during the extraction of gelatin. It has also been reported that for Type B gelatin, the viscosity is minimum and gel strength is maximum at pH 5.0 (Cole, 2000) signifying the importance of pH for its rheological properties. The pH reported for gelatin from the skin of red tilapia was 3.05 and for black tilapia it was 3.91 (Jamilah and Harvinder, 2002).

3.4.

Hydroxyproline

Hydroxyproline contents of the gelatin in the present study were in the range of 7.417.77 mg/g (Table 3), which were lesser than the gelatin extracted from tilapia skin, 8.44 mg/g (Cho et al., 2006) and cod skin, 8.30 mg/gm (Gomez-Guillen et al., 2002). Gelatin with high levels of imino acids tends to have high gel strength and melting point (Haug et al., 2004; Muyonga et al., 2004), as imino acids are important in the renaturation of gelatin subunits during gelling (JohnstonBanks, 1990).

3.5.

Gel strength (bloom value)

3.2.

Gelatin yield

Gelatin yield was found high in Tiger toothed croaker compared to Pink perch and in both the shes skins yielded higher values compared to bones. The Tiger-toothed croaker and Pink perch skins yielded 7.56% and 5.57% gelatin, while their bones yielded 4.57% and 3.55% respectively (Table 2). The yields of gelatin have been reported to vary among the sh species mainly due to differences in collagen content, the compositions of skin as well as the skin matrix. Variations in the yield have also been reported due to the differences in the diverse extraction methods followed (Gomez-Guillen et al., 2002; Jamilah and Harvinder, 2002; Muyonga et al., 2004; Jongjareonrak et al., 2006). Leaching of collagen during washing and treatments of skin could result in the lower yield of gelatin. Insufcient denaturation of soluble collagen during the extraction can also result in lower yield. The maximum swelling of the skins and bones of Tiger-toothed croaker was observed during pretreatment with alkali and acid correlating with better yield due to the opening of cross links during

Gelatin is highly capable of forming hydrogen bonds with water molecules to form a stable three-dimensional gel. The need to evaluate the characteristics of the gel has resulted in the concept of gel strength which is also known as bloom value. Gel strengths of gelatins from Tiger-toothed croaker skin/bone and Pink perch skin/bone were shown in Table 4. In both the shes, the gel strengths of bone gelatins were signicantly (p < 0.05) lower than that of skin gelatin. The gel strengths obtained in this study were similar to that of Tilapia (180.76 g) (Jamilah and Harvinder, 2002), sin croaker (124.94 g) and short n scad (176.92 g) (Cheow et al., 2007) and lower than that of Nile perch (229 g) (Muyonga et al., 2004), yellow n tuna (426 g) (Cho et al., 2005), Tilapia (263 g) (Grossman and Bergman, 1992) and Grass carp (267 g) (Kasankala et al., 2007). The ability to form weak gels may nd new applications for sh gelatin as non-gelling gelatins and it could possibly be used in refrigerated products and in products where low gelling temperatures are required (Gudmundsson, 2002). The gel strength of sh gelatin has been reported in a wide range 124426 g, compared to 200300 g for bovine or porcine gelatin (Karim and Bhat, 2009). The differences in gel strength among the various species could be explained by differences in extraction process used and the intrinsic properties of collagen which varies among sh species. Gudmundsson and Hafsteinsson (1997) suggested that the gel strength may depend on isoelectric point and may be controlled, to certain extent, by adjusting the pH.

3.6.

Viscosity

Viscosity is the second most important commercial property of gelatin after gel strength (Ward and Courts, 1977). The viscosity for the samples was in the range of 6.810.53 cP. The

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Table 1 Proximate composition of raw materials used for gelatin extraction. Source of raw materials
Tiger-toothed croaker (Otolithes ruber) Pink perch (Nemipterus japonicus) Skin Bone Skin Bone

Moisture (%)
79.50 78.63 79.63 74.83 2.84 0.03 2.07 0.58

Protein (%)
17.50 14.60 16.76 14.70 0.81 0.73 0.30 1.30

Fat (%)
1.51 1.38 1.41 1.25 0.20 0.05 0.11 0.10

Ash (%)
1.78 1.88 1.29 1.56 0.79 0.03 0.06 0.27

Values are given as mean SD from triplicate determinations.

Table 2 Proximate composition of extracted gelatin. Source of raw materials

Tiger-toothed croaker (Otolithes ruber) Pink perch (Nemipterus japonicus) Skin Bone Skin Bone

Moisture (%)
9.60 10.33 8.73 8.56 0.72 0.85 1.20 0.55

Protein (%)
86.45 82.50 72.63 69.49 0.53 0.91 1.01 0.91

Fat (%)
0.85 0.52 0.55 0.32 0.10 0.11 0.10 0.06

Ash (%)
1.88 2.70 0.30 2.80 0.14 0.17 0.01 0.10

Values are given as SD from triplicate determinations; values in the same column with different superscript differed signicantly (p < 0.05).

Table 3 Gelatin yield and its hydroxy proline content and pH. Source of raw materials
Tiger-toothed croaker (Otolithes ruber) Pink perch (Nemipterus japonicus) Skin Bone Skin Bone

Yield (%)
7.56 4.57 5.57 3.55 0.09 0.08b 0.15c 0.04d
a

Hydroxy proline (mg/g)

7.77 7.51 7.63 7.41 0.02 0.04 0.02 0.02

pH of 1% solution
4.62 4.80 4.60 4.70 0.02a 0.05b 0.00a 0.05ab

Values are given as SD from triplicate determinations; values in the same column with different superscript differed signicantly (p < 0.05).

Table 4 Rheological properties of gelatin extracted from croaker and perch. Source of gelatin
Tiger-toothed croaker (Otolithes ruber) Pink perch (Nemipterus japonicus) Skin Bone Skin Bone

Bloom Value (g)

170 150 140 130 2.88c 4.04b 2.88ab 4.40a

Viscosity (cP)
10.53 8.30 8.47 6.8 0.46c 0.26b 0.60b 0.20a

Melting point ( C)
20.36 19.5 19.23 19.0 0.29b 0.40ab 0.26a 0.17a

Values are given as standard error from triplicate determinations; values in the same column with different superscript differed signicantly.

viscosity was signicantly (p < 0.05) higher in Tiger-toothed croaker skin gelatin compared to that of Pink perch skin gelatin (Table 4). Viscosity is partially controlled by molecular weight and molecular size distribution (Sperling, 1985). The viscosities of most of the commercial gelatins have been reported up to 13.0 cP (Johnston-Banks, 1990). Jamilah and Harvinder (2002) reported the viscosity values of 3.2 cP and 7.12 cP for red and black tilapia respectively, whereas for channel cat sh it was 3.23 cP (Yang et al., 2007). The changes in pH are known to inuence the viscosity and minimum viscosity for gelatin has been observed in the pH range of 68 (Stainsby, 1987).

(Gomez-Guillen et al., 2002) and hoki (16.6 C) (Mohtar et al., 2010). However, these melting points were lower than that of Black tilapia (28.9 C) (Jamilah and Harvinder, 2002) which was warm water sh. Fish gelatin with lower melting temperature had a better release of aroma and offered stronger avour and useful in product development to control the texture and avour release during mastication. Choi and Regenstein (2000) reported that melting point increases with the maturation time and it has been observed that the levels of imino acids (proline and hydroxyl proline) contribute to the melting point characteristics (Gudmundsson, 2002).

3.8. 3.7. Melting point

Emulsifying capacity and stability

Melting point of gelatins extracted from Tiger-toothed croaker and Pink perch skins were 20.36 C and 19.23 C, while bones were 19.5 C and 19.0 C respectively. The melting point of Tiger-toothed croaker skin gelatin was signicantly higher (p < 0.05) compared to that of Pink perch (Table 4). It is known that sh gelatin has lower melting point than mammalian gelatin (Norland, 1990). The melting points of bovine gelatin and porcine gelatin have been reported as 29.7 and 32.3 C respectively (Gudmundsson, 2002). The melting points observed in the present study are far higher than those reported for cold water shes such as cod (13.8 C), hake (14 C)

Emulsiers are surface active materials that adsorb to interfaces and facilitate the production of small droplets by lowering the interfacial tension during homogenization (Walstra, 2003). The amphoteric nature with the hydrophobic zones on the peptide chain make gelatin to behave as an emulsier and it is being used in the manufacture of toffees and water-in-oil emulsions such as low fat margarine, salad dressings, and whipped cream (Baziwane and He, 2003). Emulsifying capacity and stability of gelatin are shown in Table 5. Emulsifying capacity of Tiger-toothed croaker skin and bone were 55.70% and 40.50%, while those of Pink perch were 47.50% and 35.50%. There is a growing trend within food industry

Please cite this article in press as: Koli, J.M., et al., Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus). Food Bioprod Process (2011), doi:10.1016/j.fbp.2011.08.001

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Table 5 Functional properties of extracted gelatin from croaker and perch. Source of gelatin
Tiger-toothed croaker (Otolithes ruber) Pink perch (Nemipterus japonicus) Skin Bone Skin Bone

Emulsifying capacity (%)

55.70 40.50 47.50 35.50 0.76d 0.64b 0.51c 0.57a

Emulsifying stability (%)

35.70 32.50 32.40 25.00 1.87b 0.51b 0.60b 1.50a

Foaming capacity (%)

17.00 15.00 15.00 13.00 1.60b 1.04ab 0.76ab 0.76a

Foaming stability (%)

14.00 10.00 10.00 8.00 1.96b 0.65a 0.51a 0.29a

Water holding capacity (ml/g)

4.90 3.00 2.36 1.51 0.56c 0.05b 0.06ab 0.18a

Values are given as standard error from triplicate determinations; values in the same column with different superscript differed signicantly (p < 0.05).

Table 6 Gelatin colour and gel clarity. Source of gelatin

Tiger-toothed croaker (Otolithes ruber) Pink Perch (Nemipterus japonicus) Skin Bone Skin Bone

Lightness L*
75.41 0.43 65.44 0.43b 71.74 0.13c 62.50 0.63d
a

Redness a*
2.79 1.65 2.74 1.97 0.28 0.05b 0.02c 0.60d
a

Yellowness b*
19.25 22.50 22.07 22.60 0.26 0.51b 0.05c 0.50d
a

Transmittance (%)
49.43 0.08c 40.50 1.05a 44.30 0.20b 40.13 0.30a

Values are given as standard error from triplicate determinations; values in same column with different superscript differed signicantly (p < 0.05).

to replace synthetic emulsiers with more natural sources (Garti, 1999). Proteins extracted from a verity of natural sources can be used as emulsiers in foods because of their ability to facilitate the formation, improve the stability, and produce desirable physico-chemical properties (Dickinson, 2003; McClements, 2004). Emulsifying stability (ES) was more in Tiger-toothed croaker skin and bone gelatin as compared to Pink perch skin and bone gelatin (Table 5). The functional properties of gelatin depend on several factors including the method of preparation and the intrinsic characteristics of collagen (Badii and Howell, 2006).

capacity of solubilized gelatin makes it suitable material for reducing drip loss and impairing juiciness in frozen sh or meat products when thawed or cooked, and where denatured protein has suffered a partial loss of its water-holding capacity.

3.11.

Gelatin colour and gel clarity

3.9.

Foaming capacity and stability

Foam capacity and foam stability of gelatin are shown in Table 5. The foam capacity of gelatin extracted from Tigertoothed croaker skin (17%) was higher than that from Pink perch skin (15%). The bone gelatins also followed the same pattern of skin gelatin. Foam formation is generally controlled by transportation, penetration and reorganization of protein molecules at the air-water interface. A protein must be capable of migrating rapidly to the air-water interface, unfolding and rearranging at the interface to express good foaming properties (Halling, 1981). The positive correlation between hydrophobicity of unfolded proteins and foaming characteristics has been reported (Townsend and Nakai, 1983). Foam stability depends on the nature of the lm and indicates the extent of protein interaction within the matrix (Mutilangi et al., 1996)

Colour of gelatin extracted from Tiger-toothed croaker and Pink perch was expressed in terms of L*, a*, and b* and there were signicant differences in colour characteristics among the samples studied (Table 6). Tiger-toothed croaker skin gelatin showed the greatest lightness value (L*) and the gelatins from skin samples had higher values compared to bone gelatins. Similar results were found for redness (a*) and there was no signicant difference with respect to yellowness (b*). It can be concluded that factors such as sh species and raw material inuence the colour characteristics of extracted gelatin. Both colour and clarity of a gelatin gel are important aesthetic properties, depending on the application for which the gelatin is intended. While croaker skin gelatin solution showed the highest transmittance (%T), the bone gelatin solutions of both Tiger-toothed croaker and Pink perch exhibited very poor %T (Table 6). The turbidity and dark colour of gelatin is commonly caused by inorganic, protein and mucosubstance contaminants, introduced or not removed during its extraction (Eastoe and Leach, 1977).

4.

Conclusions

3.10.

Water holding capacity

The functional properties of proteins in a food system depend in part on the water holding capacity (WHC) which refers to the ability of protein to imbibe water and retain it against a gravitational force within protein matrix. The water holding capacities of different skin and bone gelatins are given in Table 5. WHC of Tiger-toothed croaker skin and bone were higher as compared with Pink perch skin and bone gelatin. The WHC of Tiger-toothed croaker skin and bone gelatin were found 4.50 ml/g and 3.00 ml/g, while Pink perch skin and bone gelatin were 2.36 ml/g and 1.50 ml/g. The water binding

It can be concluded that sh waste from surimi processing industry may be utilized to produce gelatin. Results from the present study clearly demonstrate that compared to bones, skin yields higher amounts of gelatin having better rheological and functional properties. The skin of Tiger-toothed croaker is a prospective source to produce gelatin in good yield with the desirable characteristics comparable to commercially available mammalian gelatins.

Acknowledgement
The rst author would like to acknowledge Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli and College of

Please cite this article in press as: Koli, J.M., et al., Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus). Food Bioprod Process (2011), doi:10.1016/j.fbp.2011.08.001

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Fisheries, Shirgaon, Ratnagiri for granting the study leave for Ph. D. programme. Authors wish to express their thanks to Ulka seafood Pvt. Ltd. Taloja, Mumbai, for providing raw materials.

References
AOAC, 1995. Ofcial Methods of Analysis, 16th ed. Association of Ofcial Analytical Chemists, Washington, DC. Arnesen, J.A., Gildberg, A., 2006. Extraction of muscle proteins and gelatin from cod head. Process Biochem. 41, 697700. Asher, D.M., 1999. The transmissible spongiform encephalopathy agents: concerns and responses of the United States regulatory agencies in maintaining the safety of biologics. Dev. Biol. Standard. 100, 103118. Avena-Bustillos, R.J., Olsen, C.W., Olson, D.A., Chiou, B., Yee, E., Bechtel, P.J., McHugh, T.H., 2006. Water vapor permeability of mammalian and sh gelatin lms. J. Food Sci. 71, E202E207. Badii, F., Howell, N.K., 2006. Fish gelatin: structure, gelling properties and interaction with egg albumen proteins. Food Hydrocolloid. 20, 630640. Bergman, I., Loxley, R., 1963. Two improved and simplied methods for the spectrophotometric determination of hydroxyproline. Anal. Chem. 35 (12), 19611965. Baziwane, D., He, Q., 2003. Gelatin: the paramount food additive. Food Rev. Int. 19 (4), 423435. BSI, 1975. Methods for Sampling and Testing Gelatin (Physical and Chemical Methods). British Standard Institution, BS 757, London. Burke, C.J., Hsu, T.A., Volkin, D.B., 1999. Formulation, stability, and delivery of live attenuated vaccines for human use. Crit. Rev. Therap. Drug Carrier Syst. 16 (1), 183. Cheow, C.S., Norizah, M.S., Kyaw, Z.Y., Howell, N.K., 2007. Preparation and characterization of gelatins from the skins of sin croaker (Johnius dussumieri) and shortn scad (Decapterus macrosoma). Food Chem. 101, 386391. Cho, S.H., Jahncke, M.L., Chin, K.B., Eun, J.B., 2006. The effect of processing conditions on properties of gelatin from Skate (Raja kenojei) skins. Food Hydrocolloid 20, 810816. Cho, S.M., Gu, Y.S., Kim, S.B., 2005. Extracting optimization and physical properties of yellown tuna (Thunnus albacares) skin gelatin compared to mammalian gelatins. Food Hydrocolloid. 19, 221229. Choi, S.-S., Regenstein, J.M., 2000. Physicochemical and sensory characteristics of sh gelatin. J. Food Sci. 65, 194199. Cole, C.G.B., 2000. Gelatin. In: Frederick, J.F. (Ed.), Encyclopedia of Food Science and Technology. , 2nd ed. Wiley, New York, pp. 11831188. Dickinson, E., 2003. Hydrocolloids at interfaces and the inuence on the properties of dispersed systems. Food Hydrocolloid. 17 (1), 2539. Diniz, F.M., Martin, A.M., 1997. Effects of the extent of enzymatic hydrolysis on the functional properties of shark protein hydrolysate. Lebensmittel-Wissenschaft und-Technologie 30, 266272. Eastoe, J.E., Leach, A.A., 1977. Chemical constitution of gelatin. In: Ward, A.G., Courts, A. (Eds.), The Science and Technology of Gelatin. Academic Press, London, pp. 73107. Foegeding, E.A., Lanier, T.C., Hultin, H.O., 1996. Characteristics of edible muscle tissues. In: Fennema, O.R. (Ed.), Food Chemistry. Marcel Dekker, New York, pp. 880942. Gans, A.M., 2007. Method for treating wounds to promote healing, United States Patent: 7,682,624. Garti, N., 1999. What can nature offer from an emulsier point of view: trends and progress? Colloids Surf. A. 152 (12), 125146. Gilsenan, P.M., Ross-Murphy, S.B., 1999. Structure and rheology of gelatin gels. In: Stokke, B.T., Elgsaeter, A. (Eds.), The Wiley Polymer Networks Group Review Series, vol. 2. Wiley, Chichester, pp. 237273. GME, 2005, Standard Methods for the Testing of Edible Gelatine, Gelatine Monograph, Gelatin Manufacturers of Europe.

Gomez-Guillen, M.C., Turnay, J., Fernandez-Diaz.F M.D., Ulmo, N., Lizarbe, M.A., Montero, P., 2002. Structural and physical properties of gelatin extracted from different marine species: a comparative study. Food Hydrocolloid. 16, 2534. Grossman, S., Bergman, M., 1992. Process for the production of gelatin from sh skins. US patent: 5,093,474. Gudmundsson, M., Hafsteinsson, H., 1997. Gelatin from cod skins as affected by chemical treatments. J. Food Sci. 62, 3747. Gudmundsson, M., 2002. Rheological properties of sh gelatin. J. Food Sci. 67, 21722176. Halling, P.J., 1981. Protein stabilized foams and emulsions. CRC Crit. Rev. Food Sci. 15 (2), 155203. Haug, I.J., Draget, K.I., Smidsrod, O., 2004. Physical and rheological properties of sh gelatin compared to mammalian gelatin. Food Hydrocolloid. 18, 203213. Holzer, D., 1996. Gelatin production. US patent: 5,484,888. Jamilah, B., Harvinder, K.G., 2002. Properties of gelatins from skins of shblack tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica). Food Chem. 77 (1), 8184. Johnston-Banks, F.A., 1990. Gelatin. In: Harris, P. (Ed.), Food Gels. Elsevier Applied Science Series, London, pp. 233289. Jones, N.R., 1977. Uses of gelatin in edible products. In: Ward, A.G., Courts, A. (Eds.), The Science and Technology of Gelatin. Academic Press, London, pp. 336394. Jongjareonrak, A., Benjakul, S., Visessanguan, W., Tanaka, M., 2006. Skin gelatin from bigeye snapper and brownstripe red snapper: chemical compositions and effect of microbial transglutaminase on gel properties. Food Hydrocolloid. 20, 12161222. Karim, A.A., Bhat, R., 2009. Fish gelatins: properties, challenges, and prospects as alternative to mammalian gelatins. Food Hydrocolloid. 23, 563576. Kasankala, L.M., Xue, Y., Weilong, Y., Hong, S.D., He, Q., 2007. Optimization of gelatine extraction from grass carp (Ctenopharyngodon idella) sh skin by response surface methodology. Bioresour. Technol. 98, 33383343. Kelleher, K., 2005. Discards in the Worlds Marine Fisheries. An Update. FAO Fisheries Technical Paper No. 470, pp. 131, Rome. McClements, D.J., 2004. Protein-stabilized emulsions. Curr. Opin. Colloid Interface Sci. 9 (5), 305313. McCormick, R., 1987. Exploiting the novel properties of protein and gelatin gels. Prepared Foods 5, 204205. Miller, R., Goninger, H.S., 1976. Functional properties of enzyme modied acylated sh protein derivatives. J. Food Sci. 41 (2), 268272. Mohtar, N.F., Conrad, P., Siew-Young, Q., 2010. Optimization of gelatin extraction from hoki (Macruronus novaezelandiae) skins and measurement of gel strength and SDS-PAGE. Food Chem. 122, 307313. Morrissey, M.T., Lin, J., Ismond, A., 2005. Waste management and by-product utilization. In: Park, J.W. (Ed.), Surimi and Surimi Seafood. , 2nd ed. CRC Press, Taylor and Francis Group, Florida, pp. 279323. Mutilangi, W.A.M., Panyam, D., Kilara, A., 1996. Functional properties of properties of hydrolysates from proteolysis of heat-denatured whey protein isolate. J. Food Sci. 61 (2), 270274. Muyonga, J.H., Cole, C.G.B., Duodu, K.G., 2004. Extraction and physico-chemical characterization of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloid. 18, 581592. Nagai, T., Suzuki, N., 2000. Isolation of collagen from sh waste materialskin, bone and ns. Food Chem. 68 (3), 277281. Norland, R.E., 1990. Fish gelatin. In: Voight, M.N., Botta, J.K. (Eds.), Advances in Fisheries Technology and Biotechnology for Increased Protability. Technomic Publishing Co., Lancaster, pp. 325333. Pollack, S.V., 1990. Silicone, bre, and collagen implantation for facial lines and wrinkles. J. Dermatol. Surg. Oncol. 16 (10), 957961. Rao, K.P., 1995. Recent developments of collagen based materials for medical applications and drug delivery systems. J. Biomater. Sci.: Polym. Ed. 7 (7), 623645.

Please cite this article in press as: Koli, J.M., et al., Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus). Food Bioprod Process (2011), doi:10.1016/j.fbp.2011.08.001

FBP-263; No. of Pages 8

ARTICLE IN PRESS
food and bioproducts processing x x x ( 2 0 1 1 ) xxxxxx

Saddler, J.M., Horsey, P.J., 1987. The new generation gelatins. A review of their history, manufacture and properties. Anesthesia 42, 9981004. Schrieber, R., Gareis, H., 2007. Gelatine Handbook. Wiley-VCH, GmbH and Co, Weinheim. Sperling, L.H., 1985. Introduction of Physical Polymer Science. John Wiley, New York. Stainsby, G., 1987. Gelatin gels. In: Pearson, A.M., Dutson, T.R., Baily, A.J. (Eds.), Collagen as Food: Advances in Meat Research, vol. 4. Van Nostrand Reinhold, New York, pp. 209222. Townsend, A.A., Nakai, S., 1983. Relationships between hydrophobicity and foaming characteristics of food proteins. J. Food Sci. 48, 588594. Vannuccini, S., 2004. Fish utilization. In: Wijkstorm, U., Gumy, A., Graigner, R. (Eds.), The State of World Fisheries and Aquaculture. Food and Agriculture Organization of the United Nations, Rome, Italy, pp. 5658. Wainewright, F.W., 1977. Physical tests for gelatin and gelatin products. In: Ward, A.G., Courts, A. (Eds.), The Science and

Technology of Gelatins. Academic Press Inc., London, pp. 508531. Walstra, P., 2003. Physical Chemistry of Foods. Marcel Dekker, New York. Ward, A.G., Courts, A., 1977. The Science and Technology of Gelatin. Academic Press Inc., London. Wasswa, J., Tang, J., Gu, X., 2007. Utilization of sh processing by products in the gelatin industry. Food Rev. Int. 23 (2), 159174. Wilesmith, J.W., Ryan, J.B., Atkinson, M.J., 1991. Bovine spongiform encephalopathy: epidemiological studies on the origin. Vet. Rec. 128 (9), 199203. Yang, H., Wang, Y., Jiang, M., Oh, J.H., Herring, J., Zhou, P., 2007. 2-step optimization of the extraction and subsequent physical properties of Channel Catsh (Ictalurus punctatus) skin gelatin. J. Food Sci. 72 (4), 188195. Yasumatsu, K., Sawada, K., Moritaka, S., Misaki, M., Toda, J., Wada, T., Ishii, K., 1972. Whipping and emulsifying properties of soybean products. Agric. Biol. Chem. 36, 719726.

Please cite this article in press as: Koli, J.M., et al., Functional characteristics of gelatin extracted from skin and bone of Tiger-toothed croaker (Otolithes ruber) and Pink perch (Nemipterus japonicus). Food Bioprod Process (2011), doi:10.1016/j.fbp.2011.08.001