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Gelatins from three cultured freshwater fish skins obtained by liming process
Food Hydrocolloids 25 (2011) 1256e1260
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Gelatins from three cultured freshwater fish skins obtained by liming process B. Jamilah a, *, K.W. Tan a, M.R. Umi Hartina b, A. Azizah a a b
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Halal Products and Research Institute, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 September 2009 Accepted 28 November 2010
The physico-chemical properties of gelatins from the skins of Red tilapia (Oreochromis nilotica), Walking catfish (Clarias batrachus) and Striped catfish (Pangasius sutchi fowler) obtained through a liming process for 14 days were evaluated. All the gelatins had very mild to undetectable fishy odour and had acceptable colour attributes, which were light yellowish to whitish. The highest gelatin yield (dry basis) was obtained from red tilapia (39.97%) skin and the bloom strength exceeded 300 g. The pH values of the gelatins were in the vicinity of 5.0. The viscosity (cp) was highest in striped catfish, followed by red tilapia and walking catfish. Their melting points were in the vicinity of 26 1 C. Turbidity was lowest in the red tilapia gelatin. Glycine, proline and alanine were the three highest amino acids found in all the gelatins obtained. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Freshwater fish skin Gelatin Liming Physico-chemical properties
1. Introduction In 2005, the total gelatin production, which is expected to increase, was in the range of approximately 305,000 metric tons (Schrieber & Gareis, 2007). The gelatins are mainly produced from the skins, hides and bones of bovine and porcine. However, the production of fish gelatin only stands at 1.5% in 2007 (GME Market data, 2007), even though about 30% of the fish processing wastes comprise of skin, scale and bone which are very rich in collagen, the precursor of gelatin (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005). Kolodziejska, Skierka, Sadowska, Kolodziejski, and Niecikowska (2008) recently reported on the extraction of gelatin from offals of Baltic cod, cold-smoked salmon and herring. However, on the overall, the utilization of these industrial wastes has not been exhaustively exploited. The versatility of application of fish gelatin as a functional food ingredient depends very much on its properties and should not be viewed as an inferior substitute of the traditional gelatin. The major physical properties of gelatin are gel strength and melting point, which are governed mainly by the amino acid composition (pro þ hyp content), molecular weight distribution and also the ratio of a/b chains contained in the gelatin (Karim & Bhat, 2009). The amino acid content in a gelatin is dependent on the origin of the raw material; however, the overall property is also influenced by the production method employed. Gelatin from cold-water fish
* Corresponding author. Tel.: þ603 89468396; fax: þ603 89423552. E-mail address: [email protected] (B. Jamilah). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.11.023
species has weaker gelling properties due to the low content of proline and hydroxyproline, as compared to the bovine and porcine derived gelatins. Such gelatins have been reported to have a good film formation and emulsifying properties (Schrieber & Gareis, 2007). Karim and Bhat (2009) also suggested that gelatin with low melting point could be used in dry products for microencapsulation. Gelatin from several fish species have been studied, including warm-water fish; megrim (Lepidorhombus boscii) (Gómez-Guillén & Montero, 2001); tilapia (Oreochromis nilotica) (Jamilah & Harvinder, 2002); Nile perch (Lates niloticus) (Muyonga, Cole, & Duodu, 2004); channel catfish (Ictalurus punctatus) (Yang, Wang, Zhou, & Regenstein, 2008) and cold-water fish; Baltic cod (Godus morhua) (Kolodziejska, Kaczorowski, Piotrowska, & Sadowska, 2004); Alaska Pollock (Theragra chalcogramma) (Zhou & Regenstein, 2005); Atlantic salmon (Salmo solar) (Arnesen & Gildberg, 2007). Recent study on gelatins obtained from four Malaysian marine species, has been reported (Irwandi et al., 2009). Fish gelatins are normally produced through acid extraction method reported properties typically known for the similar gelatin group. Reports on liming process for fish gelatin extraction are very limited. Liming process is particularly designed for gelatin extraction from mammalian skins and bones, which normally takes a few days to four months, depending on the concentration and temperature used (Schrieber & Gareis, 2007). However, in 2002, an improved alkaline process for preparing type B fish gelatin was patented by Stanley (2002). The process described consisted of an initial extraction step by liming for 42 days, which may be followed by an acid extraction process. Lime [Ca(OH)2] is normally preferred
B. Jamilah et al. / Food Hydrocolloids 25 (2011) 1256e1260
due to its ability to regulate the desired alkalinity and does not cause the collagen to swell (Ockermann & Hansen, 1988). In addition, the yield and Bloom strength of gelatin are often elevated by liming method at optimum extraction conditions. The quality of the gelatin with respect to Bloom strength and viscosity can be a result of the relationship between the concentration of alkaline solution, temperature and duration of conditioning. Stronger liming conditioning normally results in higher viscosity (Schrieber & Gareis, 2007). Red tilapia, walking catfish and striped catfish have worldwide recognition as the most important warm-water cultured fish commodity. They are abundantly found in the ASIAN region, the African continent and other parts of the world such as Central America. Since liming process has not been reported for the extraction of gelatin from red tilapia (O. nilotica), walking catfish (Clarias batrachus) and striped catfish (Pangasius sutchi fowler), therefore, this study was carried out to evaluate the potential of the process. The physico-chemical characteristics of the obtained gelatin were used as indicators of the suitability of the process.
2. Materials and methods 2.1. Materials Live red tilapia (O. nilotica) (500e600 g), walking catfish (C. batrachus) (200e250 g) and striped catfish (P. sutchi fowler) (400e500 g) bought from a local farm. Upon arrival in the laboratory, they were immediately killed, filleted and the skins were collected. The skins were weighed, packed in polyethylene bags and freeze-stored at 20 C if not used immediately. All reagents used in the studies were of analytical grade.
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2.3.2. Proximate composition The moisture, crude protein (micro Kjedahl) and ash content of fish skins and gelatin were determined according to AOAC (1995) standard procedures. 2.3.3. Visual observations of colour Visual observation for colour and odour characteristic were noted as reported by Jamilah and Harvinder (2002) which was an adaptation of Gudmundsson and Hafsteinsson (1997) 2.3.4. Odour by sensory evaluation The evaluation was also carried out according to Gudmundsson and Hafsteinsson (1997). The odour of the dried gelatin and gelatin solution was evaluated by judging the intensity of fishy odour. Odour of dry gelatin and gelatin solutions was evaluated by 10 trained panelists. The different experimental runs were evaluated for odour and colour and informal comparison to fish gelatin from Norland was made. The gelatin was sniffed 3 times and the odour was noted. 2.3.5. Instrumental colour Colour measurements were made using HunterLab Ultrascan Sphere Spectrocolorimeter (model Minolta Cr-300 Series, US.). The samples were filled in a clear plastic container and three readings were taken for each sample. The colour values were expressed as ‘L’ e ‘lightness’, ‘a’ e ‘redness’ and ‘b’ e ‘yellowness’. 2.3.6. Amino acid composition The amino acids composition in gelatins was determined using Waters-Pico Tag Amino Acid Analyzer High Performance Liquid Chromatography (Model: Waters 501 Millipore Corporation, USA) with Waters column measuring 3.9 150 mm. Each sample was hydrolyzed with 6 N HCl at 110 C for 24 h. The hydrolysis was analyzed for their free amino acid content on a Waters auto analyzer, as recommended in the Waters-501 Instruments Manual (1991). Readings were carried out in triplicates.
2.2. Gelatin extraction Skins were washed under running tap water to remove superfluous materials. The wash skins were drip-dried and soaked in the saturated lime solution [Ca(OH)2] at the concentration of 27 g L1, at 20 C for 14 days (Holzer, 1996). For each kilogram of wet skins, 2 L of Ca(OH)2 solution was used as the soaking medium. After soaking, the skins were then removed and washed with abundant tap water (1:10) to remove excessive Ca(OH)2 while maintaining the skins at pH 10. This was followed by soaking in distilled water at 48 C overnight to solubilise the gelatin. The solution was then filtered through Whatman No. 4 and 541 filter papers before passing through a strong acid cationic exchange resin, which reduced the pH of gelatin to approximately 5. The filtrates were then freeze-dried and further analyzed for physico-chemical properties.
2.3. Analyses 2.3.1. Yield of gelatin The yields of the gelatins obtained were calculated on both weight and protein basis as follows: (i) % Yield (wet wt basis) ¼ (Dry wt of gelatin/wet wt of skin) 100% (ii) % Yield (dry wt basis) ¼ (Dry wt of gelatin)/(wet wt of skin moisture content) 100% (iii) % Yield (protein basis) ¼ (Dry wt of gelatin/protein content of skin) 100%
2.3.7. Determination of melting point The melting point of gelatin samples was determined according to Choi and Regenstein (2000). Melting point determination was carried out in triplicates. 2.3.8. Determination of viscosity The viscosity of the gelatin (6.67% concentration at 60 C) was performed in a cone-plate cell of the Brookfield DV III Rheometer (model RV, USA). The adapter and spindle used to measure the viscosity was SC 4-18. The speed of the spindle was adjusted to 60 0.5 rpm. Data obtained were the average of five determinations and were expressed as centipoises (cp). 2.3.9. Determination of turbidity The turbidity of 6.67% gelatin solution (w/v) which was prepared with distilled water was determined using a turbidimeter (2100P HACH 46500-00, HACH Company, Colorado, US). Readings were carried out in triplicates for all samples. 2.3.10. Determination of isoelectric point The isoelectric points of the extracted gelatins were determined by running the isoelectric focusing using Bio-Rad PROTEAN IEF Cell (Bio-Rad Laboratories Inc., Richmond, CA, USA) with the isoelectrofocusing ReadyStrip IPG Strips pH range 3e10 (Bio-Rad). The runs for each gelatin were carried at least 3 times. 2.3.11. Determination of gel strength and pH The gel strength and the pH of gelatins were measured according to the method recommended by the manufacturer of the
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Table 1 Yield of gelatins from three selected freshwater fishes. Types of fish
Red tilapia Walking catfish Striped catfish a
Yielda Wet basis (%)
Dry basis (%)
Protein basis (%)
12.92 13.06 11.17
39.97 32.06 26.23
44.44 42.12 33.15
Values were means of readings from two separate batches.
texture measuring instrument. The gel strength was determined using TA-XT2 Texture Analyzer Stable Micro Systems (England) with 0.500 Radius Cylinder Probe (P/0.5R) as recommended by the manufacturer. A gelatin solution of 6.67% (w/v) was prepared with distilled water and transferred into a standard Bloom jar. The gelatins were left to mature for 17 1 h at 10 C in a water bath. After maturation period, the jars were removed immediately from the water bath and placed centrally under the probe and the penetration test was commenced. The load cell used was 5 kN. The unit for the response was recorded as grams (g). The pH was measured with a glass electrode (Toledo 320 pH Meter, Mettler-Toledo Instrument, Greifensee, Switzerland) after standardizing with pH 4.0 and 7.0 buffers. The gel strength and pH determination were carried out in triplicates. 2.3.12. Statistical analysis All data collected were analysed using Analysis of Variance (ANOVA) and Duncan’s Multiple Test to determine the significance level among the means using the statistical programme analyses package (SAS, 1989). 3. Results and discussion 3.1. Yield of gelatin The recovery of gelatin based on wet, dry and protein basis is as shown in Table 1. It was observed that the extractability of gelatin among the three species varies. The highest yield was obtained from red tilapia skin (dry weight basis). The result was higher than that reported by Jamilah and Harvinder (2002) for acid extracted gelatin but lower than those reported by Grossman and Bergman (1992) and Holzer (1996) for the same fish species. The yields of gelatin (protein basis) from walking and striped catfish skins were higher than reported by Yang et al. (2008) for channel catfish skin, which used NaOH for the alkaline pretreatment. This suggested that the use of CaOH2 to condition the skin prior to gelatin extraction resulted in better recovery. 3.2. Proximate composition of fish skins and gelatin Table 2 shows the protein, moisture and ash content of the skins of the three selected freshwater fish skins and the extracted gelatins. The skin of red tilapia skin has the highest moisture content, followed by that of walking and striped catfish. All the three freshwater fish skins had less ash contents (0.46e0.51%) compared
to that of the Nile perch skin (3.7%) (Muyonga et al., 2004). They also had higher protein contents compared to that of Nile perch reported by Muyonga et al. (2004). The maximum possible yield of gelatin to be extracted from the skin is related to the protein content of the collagenous material in the skin although it is not a direct indication. The crude protein content of the skin is therefore not a good indicator of the possible protein content of the gelatin obtained in this procedure. Of late, the composition of hydroxyproline (hyp) in the collagenous material has been suggested as a better indicator to determine the yield of gelatin extracted from fish skin (Nalinanon, Benjakul, Visessanguan, & Kishimura, 2008). However, the amino acid analytical procedure used in this experiment did not allow the detection of hydroxyproline. The ash content of all the gelatins obtained was comparatively low (0.08e0.18%). This was probably due to the ionization process that reduced the mineral content. Gelatin from skate (Raja kenojei) fish skin, which was also produced by the liming process but without ionization process, had higher ash content of approximately 1.4% (Cho, Jahncke, Chin, & Eun, 2006). 3.3. Physical characteristics of the fish gelatins All the determined physical properties of the extracted gelatins were tabulated in Table 3. Great variations in the gel strengths of the gelatins obtained were observed (Table 3). The highest strength was obtained in red tilapia gelatin (384.9 g), followed by walking and striped catfish, 238.9 g and 147.4 g, respectively. They were significantly different from each other. Gelatin obtained from red tilapia skin by liming process had higher Bloom strength, which was approximately 3 times higher than that reported by Jamilah and Harvinder (2002) for gelatin obtained through acid and alkaline conditioning process (gelatin Bloom strength was 128.1 g). Therefore, it is highly possible to produce gelatin of different gel strength from the same source by merely varying the extraction method even though the gel strength has always been associated to the natural characteristics of the protein matrix of the source. Suggestions such as varying the pH and the isoelectric point closer to the natural isoelectric point of the protein to produce stiffer and more compact gels had also been put forth (Gudmundsson & Hafsteinsson, 1997). Inducing the increase in the cross-linking of the gelatin, and thus increasing the gel strength of the gelatin with the addition of 1.0 mg/g transglutaminase has been recently reported (Norziah, Hassan, Khairulnizam, Mordi, & Norita, 2009). The gel strength of both gelatins from red tilapia and striped catfish obtained by this process were comparable to the commercial gel strengths of those from bovine and porcine origin (200e240 g). Therefore, these gelatins can be used as a substitute or complementary ingredient in required applications. Slight variations in the melting points of the gelatins were noted. However, they were higher than reported for skate (R. kenojei) fish skins which were in the range of 18e21 C (Cho et al., 2006). The melting temperature of striped catfish gelatin was similar to that of gelatin from Nile perch skin (Muyonga et al., 2004).
Table 2 The proximate compositiond of the three selected freshwater fish skins and gelatins. Fish species
Red tilapia Walking catfish Striped catfish
Moisture
Protein
Ash
Skin
Gelatin
Skin
Gelatin
Skin
Gelatin
70.43 0.26a 62.47 0.34b 60.52 0.52c
8.51 0.21a 7.86 0.53ab 7.29 0.42a
29.07 0.31a 31.01 0.48b 33.70 0.75c
93.25 0.21a 77.88 0.53b 80.02 0.33c
0.51 0.09a 0.52 0.23a 0.46 0.58a
0.18 0.01a 0.26 0.30a 0.08 0.17a
Values with the different superscripts within each column were significantly different (p < 0.05). d Values were means SD from triplicate determinations.
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Table 3 The physico-chemical propertiesd of the gelatins obtained from the three selected freshwater fish. Properties
Red tilapia
Gel strength (g) Melting point ( C) Viscosity (cp) Turbidity (NTU) Isoelectric point pH at 25 C Hunter colour value ‘L’ ‘a’ ‘b’ Sensory attributes Colour appearance (powder)
384.9 27.77 7.70 176 4.2 5.38
Whitish Shiny and soft textured
Odour (powder) Odour after redissolving
Barely detectable fishy odour Slightly fishy when re-dissolved
Walking catfish
4.49a 0.25a 0.23a 6.5a 0.5a 0.02a
147.40 24.96 6.28 495 5.3 5.02
79.45 1.10a 0.71 0.09a 5.75 0.14a
12.29b 0.25b 0.83b 6.5b 0.4b 0.08b
67.37 0.53b 2.37 0.04b 7.48 0.50b Dark brownish Shiny and slightly coarse texture Emitted slightly fishy odour Detectable fishy odour
Striped catfish 238.90 26.20 8.21 511 5.1 5.12
8.54c 0.26c 0.57c 13.1c 0.2b 0.03b
68.69 0.10c 2.66 0.04c 7.98 0.24c Light brownish Shiny and medium soft texture Detectable fishy odour Detectable fishy odour
Values with the different superscripts within each row were significantly different (p < 0.05). d Values were means SD from triplicates determination.
Viscosity is the second most important commercial physical property of a gelatin (Wards & Courts, 1997), which is partially controlled by molecular weight and polydipersity of the peptides. Higher molecular weight will increase viscosity but the polydipersity can have various effects depending on the molecular weight distribution (Gudmundsson & Hafsteinsson, 1997). The viscosities of the gelatins obtained were significantly different (p < 0.05). Their viscosities values were in the range of 6e9 cp. Grossman and Bergman (1992) reported tilapia skin gelatin had a viscosity of 5.1 cp. Lower viscosity (<3.0 cp) was reported for gelatin from channel catfish (Yang et al., 2008). The above results thus indicate that natural variations in the viscosity can be expected from different freshwater fish species, although the extraction methods do play a role. Turbidity or degree of clarity of a solution is very important in food applications such as in the addition of gelatin as a thickening agent. Turbidity values (Table 3) of the gelatins extracted from the three fish species were in the range of 176e511 NTU. Red tilapia skin gelatin exhibited lower turbidity than the gelatins obtained from either striped or walking catfish. Muyonga et al. (2004) stated that efficiency of the filtration process during gelatin extraction affected the degree of turbidity of gelatin solution, which may explain the results obtained for striped and walking catfish gelatins. Table 4 Amino acids contents (mg/g) of the gelatins obtained from the three selected freshwater fishes. Amino acid (mg/g)a
Red tilapia
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Total
43.94 71.29 31.60 197.00 ND ND 42.11 20.07 123.71 5.23 15.74 15.20 ND ND 11.88 34.32 8.04 620
1.05 1.15 0.11 0.21
0.98 1.24 2.73 0.23 0.39 1.44
0.28 1.44 1.75
Walking catfish
Striped catfish
55.44 79.27 35.95 204.13 ND 75.95 21.95 88.41 122.65 7.63 21.60 20.86 ND 11.72 23.47 27.94 35.15 832
46.79 78.94 30.79 209.80 ND 79.39 36.23 96.20 136.13 6.45 23.97 22.03 ND 16.24 36.35 26.21 25.75 871
2.68 4.58 4.37 15.68 3.80 3.21 2.27 9.94 0.64 2.40 1.99 1.51 1.53 1.62 1.97
ND e not detected. a Values are presented as the mean SD of three readings.
5.42 3.71 0.70 9.32
11.35 12.26 9.48 5.31 1.11 2.44 1.47
1.54 4.67 3.84 11.02
The turbidity value of red tilapia skin gelatin was closed to that obtained for Nile perch skin gelatin extracted at 60 C (Muyonga et al., 2004). The isoelectric points of the fish gelatins were in the range of 4.2e5.3, which were typical for type B gelatin. No earlier reports could be found to compare our results. Colour characteristics were also different among the gelatins and this could be due to the effect of the carry over of pigments from their skins. The ‘L’ (lightness) values of the three gelatins are significantly different (p < 0.05) and this confirms the visual observations on the gelatins. The colour of the red tilapia gelatin obtained in this study was different from that reported by Jamilah and Harvinder (2002) for the same fish species. The reported ‘L’ value for red tilapia gelatin using acidic treatment was lighter (92.4). Fish odour in the red tilapia gelatin was barely detectable but in both of the catfish species, fishy odour was slightly stronger. The odour description of red tilapia gelatin is similar to that of Jamilah and Harvinder (2002). They suggested that stronger fishy odour is correlated to the muddy odour and flavour associated with the fish species and also perhaps related to the feed of the fish. 3.4. Amino acids composition The total amino acid concentration was highest in striped catfish, followed by walking catfish and red tilapia. The rigidity of the gelatin gel is directly related to its proline (Pro) and hydroxyproline (Hyp) contents (Holzer, 1996). In this study, Hyp was not determined due to the inability of method employed to detect its presence as mentioned earlier. Glycine and proline, were the two main amino acids which made up a quarter (w25%) of the total amino acids content (Table 4). The highest glycine and proline contents were found in the striped catfish. The composition of amino acids for sole, megrim, cod, hake and squid was reported to be similar to the composition of interstitial collagen, accounting to >30% Gly and w17% imino acids (Pro þ Hyp) (Gómez-Guillén et al., 2002). A higher amino acid content of Pro, Hyp and Ala in commercial gelatin from tilapia has been previously shown as one of the major cause for its higher viscoelastic properties than megrim gelatin (Gómez-Guillén, Sarabia, & Montero, 2000). Glutamine as the third highest amino acid in all the extracted gelatin and no obvious difference between that of walking catfish and the striped catfish. Alanine was significantly higher in walking catfish and striped catfish as compared to the red tilapia. The values were approximately 4e5 times higher to that found in the red tilapia. Both isoleucine (Ile) and arginine (Arg) were not detected in
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the tilapia gelatin. Cystine (Cys) and histidine (His) were also not detected in all extracted gelatins. 4. Conclusion This study has proven that the liming process employed is a viable process for the extraction of the gelatin from fish skins to produce higher Bloom. The characteristics of the gelatins obtained in this process indicate that they have good commercial application. The red tilapia gelatin showed superior characteristics as compared to the other two species studied. Acknowledgement The authors would like to extend their sincere thanks to the Malaysian Ministry of Science and Technology (MOSTI) for providing the Intensive Research Priority Area (IRPA) grant to carry out this study. References AOAC. (1995). Official method of analysis. Washington D.C., USA: Association of Official Chemist. Arnesen, J. A., & Gildberg, A. (2007). Extraction and characterisation of gelatine from Atlantic salmon (Salmo salar) skin. Bioresource Technology, 98, 53e57. Choi, S. S., & Regenstein, J. M. (2000). Physico-chemical and sensory characteristic of fish gelatin. Journal of Food Science, 65, 194e199. Cho, S. H., Jahncke, M. L., Chin, K. B., & Eun, J. B. (2006). The effect of processing conditions on the properties of gelatin from skate (Raja kenojei) skins. Food Hydrocolloids, 20, 810e816. GME Market data. (2007). Official website of GME. Brussels, Belgium: Gelatin Manufacturers of Europe. http://www.gelatine.org GME Market Data. Gómez-Guillén, M. C., & Montero, P. (2001). Extraction of gelatin from megrim (Lepidorhombus boscii) skins with several organic acids. Journal of Food Science, 66(2), 213e216. Gómez-Guillén, M. C., Sarabia, A. I., & Montero, P. (2000). The effect of added salts on the viscoelastic properties of fish skin gelatins. Food Chemistry, 70, 71e76. Gómez-Guillén, M. C., Turnay, J., Fernandez-Diaz, 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 Hydrocolloids, 16, 25e34. Grossman, S., Bergman, M. (1992). Process for the production of gelatin from fish skins. U.S. patent 5,093,474.
Gudmundsson, M., & Hafsteinsson, H. (1997). Gelatin from cod skins as affected by chemical treatments. Journal of Food Science, 62, 37e47. Holzer, D. (1996). Gelatin production. U.S. patent 5,484,888. Irwandi, J., Faridayanti, S., Mohamed, E. S. M., Hamzah, M. S., Torla, H. H., & Che Man, Y. B. (2009). Extraction and characterisation of gelatin from different marine fish species in Malaysia. International Food Research Journal, 16, 381e389. Jamilah, B., & Harvinder, K. G. (2002). Properties of gelatins from skins of fish-black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica. Food Chemistry, 77, 81e84. Karim, A. A., & Bhat, R. (2009). Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocolloids, 23, 563e576. Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka, M. (2005). Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chemistry, 89, 363e372. Kolodziejska, I., Kaczorowski, K., Piotrowska, B., & Sadowska, M. (2004). Modification of the properties of gelatin from skins of Baltic cod (Gadus morhua) with transglutaminase. Food Chemistry, 86, 203e209. Kolodziejska, I., Skierka, E., Sadowska, M., Kolodziejski, W., & Niecikowska, C. (2008). Effect of extracting time and temperature on yield of gelatin from different fish offal. Food Chemistry, 107, 700e706. Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004). Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloids, 18, 581e592. Nalinanon, S., Benjakul, S., Visessanguan, W., & Kishimura, H. (2008). Improvement of gelatin extraction from bigeye snapper skin using pepsin-aided process in combination with protease inhibitor. Food Hydrocolloids, 22, 615e622. Norziah, M. H., Al-Hassan, A., Khairulnizam, A. B., Mordi, M. N., & Norita, M. (2009). Characterization of fish gelatin from surimi processing wastes: thermal analysis and effect of transglutaminase on gel properties. Food Hydrocolloids, 23, 1610e1616. Ockermann, H. W., & Hansen, C. L. (1988). Glue and gelatin. In Animal by-product processing (pp. 132e157). England, Chichester: Elli Horwood Ltd. SAS. (1989). Statistical analysis system for data analysis. North Carolina, USA: The SAS Institute. Schrieber, R., & Gareis, H. (2007). Manufacture of gelatin: theory and practice. In Gelatin handbook, theory and industrial practice (pp. 42e88). Weinheim: Wiley-VCH. Stanley, F. D. (2002). Improved alkaline process for preparing type B fish gelatin. World Intellectual Property Organization. WO/2002/094959. Ward, A. G., & Courts, A. (1977). The science and technology of gelatin. London: Academic Press. Yang, H., Wang, Y., Zhou, P., & Regenstein, J. M. (2008). Effects of alkaline and acid pretreatment on the physical properties and nanostructures of the gelatin from channel catfish skins. Food Hydrocolloids, 22, 1541e1550. Zhou, P., & Regenstein, J. M. (2005). Effects of alkaline and acid pretreatments on Alaska pollock skin gelatin extraction. Journal of Food Science, 70(6), C392eC396.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Gelatins from three cultured freshwater fish skins obtained by liming process B. Jamilah a, *, K.W. Tan a, M.R. Umi Hartina b, A. Azizah a a b
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia Halal Products and Research Institute, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 September 2009 Accepted 28 November 2010
The physico-chemical properties of gelatins from the skins of Red tilapia (Oreochromis nilotica), Walking catfish (Clarias batrachus) and Striped catfish (Pangasius sutchi fowler) obtained through a liming process for 14 days were evaluated. All the gelatins had very mild to undetectable fishy odour and had acceptable colour attributes, which were light yellowish to whitish. The highest gelatin yield (dry basis) was obtained from red tilapia (39.97%) skin and the bloom strength exceeded 300 g. The pH values of the gelatins were in the vicinity of 5.0. The viscosity (cp) was highest in striped catfish, followed by red tilapia and walking catfish. Their melting points were in the vicinity of 26 1 C. Turbidity was lowest in the red tilapia gelatin. Glycine, proline and alanine were the three highest amino acids found in all the gelatins obtained. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Freshwater fish skin Gelatin Liming Physico-chemical properties
1. Introduction In 2005, the total gelatin production, which is expected to increase, was in the range of approximately 305,000 metric tons (Schrieber & Gareis, 2007). The gelatins are mainly produced from the skins, hides and bones of bovine and porcine. However, the production of fish gelatin only stands at 1.5% in 2007 (GME Market data, 2007), even though about 30% of the fish processing wastes comprise of skin, scale and bone which are very rich in collagen, the precursor of gelatin (Kittiphattanabawon, Benjakul, Visessanguan, Nagai, & Tanaka, 2005). Kolodziejska, Skierka, Sadowska, Kolodziejski, and Niecikowska (2008) recently reported on the extraction of gelatin from offals of Baltic cod, cold-smoked salmon and herring. However, on the overall, the utilization of these industrial wastes has not been exhaustively exploited. The versatility of application of fish gelatin as a functional food ingredient depends very much on its properties and should not be viewed as an inferior substitute of the traditional gelatin. The major physical properties of gelatin are gel strength and melting point, which are governed mainly by the amino acid composition (pro þ hyp content), molecular weight distribution and also the ratio of a/b chains contained in the gelatin (Karim & Bhat, 2009). The amino acid content in a gelatin is dependent on the origin of the raw material; however, the overall property is also influenced by the production method employed. Gelatin from cold-water fish
* Corresponding author. Tel.: þ603 89468396; fax: þ603 89423552. E-mail address: [email protected] (B. Jamilah). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.11.023
species has weaker gelling properties due to the low content of proline and hydroxyproline, as compared to the bovine and porcine derived gelatins. Such gelatins have been reported to have a good film formation and emulsifying properties (Schrieber & Gareis, 2007). Karim and Bhat (2009) also suggested that gelatin with low melting point could be used in dry products for microencapsulation. Gelatin from several fish species have been studied, including warm-water fish; megrim (Lepidorhombus boscii) (Gómez-Guillén & Montero, 2001); tilapia (Oreochromis nilotica) (Jamilah & Harvinder, 2002); Nile perch (Lates niloticus) (Muyonga, Cole, & Duodu, 2004); channel catfish (Ictalurus punctatus) (Yang, Wang, Zhou, & Regenstein, 2008) and cold-water fish; Baltic cod (Godus morhua) (Kolodziejska, Kaczorowski, Piotrowska, & Sadowska, 2004); Alaska Pollock (Theragra chalcogramma) (Zhou & Regenstein, 2005); Atlantic salmon (Salmo solar) (Arnesen & Gildberg, 2007). Recent study on gelatins obtained from four Malaysian marine species, has been reported (Irwandi et al., 2009). Fish gelatins are normally produced through acid extraction method reported properties typically known for the similar gelatin group. Reports on liming process for fish gelatin extraction are very limited. Liming process is particularly designed for gelatin extraction from mammalian skins and bones, which normally takes a few days to four months, depending on the concentration and temperature used (Schrieber & Gareis, 2007). However, in 2002, an improved alkaline process for preparing type B fish gelatin was patented by Stanley (2002). The process described consisted of an initial extraction step by liming for 42 days, which may be followed by an acid extraction process. Lime [Ca(OH)2] is normally preferred
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due to its ability to regulate the desired alkalinity and does not cause the collagen to swell (Ockermann & Hansen, 1988). In addition, the yield and Bloom strength of gelatin are often elevated by liming method at optimum extraction conditions. The quality of the gelatin with respect to Bloom strength and viscosity can be a result of the relationship between the concentration of alkaline solution, temperature and duration of conditioning. Stronger liming conditioning normally results in higher viscosity (Schrieber & Gareis, 2007). Red tilapia, walking catfish and striped catfish have worldwide recognition as the most important warm-water cultured fish commodity. They are abundantly found in the ASIAN region, the African continent and other parts of the world such as Central America. Since liming process has not been reported for the extraction of gelatin from red tilapia (O. nilotica), walking catfish (Clarias batrachus) and striped catfish (Pangasius sutchi fowler), therefore, this study was carried out to evaluate the potential of the process. The physico-chemical characteristics of the obtained gelatin were used as indicators of the suitability of the process.
2. Materials and methods 2.1. Materials Live red tilapia (O. nilotica) (500e600 g), walking catfish (C. batrachus) (200e250 g) and striped catfish (P. sutchi fowler) (400e500 g) bought from a local farm. Upon arrival in the laboratory, they were immediately killed, filleted and the skins were collected. The skins were weighed, packed in polyethylene bags and freeze-stored at 20 C if not used immediately. All reagents used in the studies were of analytical grade.
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2.3.2. Proximate composition The moisture, crude protein (micro Kjedahl) and ash content of fish skins and gelatin were determined according to AOAC (1995) standard procedures. 2.3.3. Visual observations of colour Visual observation for colour and odour characteristic were noted as reported by Jamilah and Harvinder (2002) which was an adaptation of Gudmundsson and Hafsteinsson (1997) 2.3.4. Odour by sensory evaluation The evaluation was also carried out according to Gudmundsson and Hafsteinsson (1997). The odour of the dried gelatin and gelatin solution was evaluated by judging the intensity of fishy odour. Odour of dry gelatin and gelatin solutions was evaluated by 10 trained panelists. The different experimental runs were evaluated for odour and colour and informal comparison to fish gelatin from Norland was made. The gelatin was sniffed 3 times and the odour was noted. 2.3.5. Instrumental colour Colour measurements were made using HunterLab Ultrascan Sphere Spectrocolorimeter (model Minolta Cr-300 Series, US.). The samples were filled in a clear plastic container and three readings were taken for each sample. The colour values were expressed as ‘L’ e ‘lightness’, ‘a’ e ‘redness’ and ‘b’ e ‘yellowness’. 2.3.6. Amino acid composition The amino acids composition in gelatins was determined using Waters-Pico Tag Amino Acid Analyzer High Performance Liquid Chromatography (Model: Waters 501 Millipore Corporation, USA) with Waters column measuring 3.9 150 mm. Each sample was hydrolyzed with 6 N HCl at 110 C for 24 h. The hydrolysis was analyzed for their free amino acid content on a Waters auto analyzer, as recommended in the Waters-501 Instruments Manual (1991). Readings were carried out in triplicates.
2.2. Gelatin extraction Skins were washed under running tap water to remove superfluous materials. The wash skins were drip-dried and soaked in the saturated lime solution [Ca(OH)2] at the concentration of 27 g L1, at 20 C for 14 days (Holzer, 1996). For each kilogram of wet skins, 2 L of Ca(OH)2 solution was used as the soaking medium. After soaking, the skins were then removed and washed with abundant tap water (1:10) to remove excessive Ca(OH)2 while maintaining the skins at pH 10. This was followed by soaking in distilled water at 48 C overnight to solubilise the gelatin. The solution was then filtered through Whatman No. 4 and 541 filter papers before passing through a strong acid cationic exchange resin, which reduced the pH of gelatin to approximately 5. The filtrates were then freeze-dried and further analyzed for physico-chemical properties.
2.3. Analyses 2.3.1. Yield of gelatin The yields of the gelatins obtained were calculated on both weight and protein basis as follows: (i) % Yield (wet wt basis) ¼ (Dry wt of gelatin/wet wt of skin) 100% (ii) % Yield (dry wt basis) ¼ (Dry wt of gelatin)/(wet wt of skin moisture content) 100% (iii) % Yield (protein basis) ¼ (Dry wt of gelatin/protein content of skin) 100%
2.3.7. Determination of melting point The melting point of gelatin samples was determined according to Choi and Regenstein (2000). Melting point determination was carried out in triplicates. 2.3.8. Determination of viscosity The viscosity of the gelatin (6.67% concentration at 60 C) was performed in a cone-plate cell of the Brookfield DV III Rheometer (model RV, USA). The adapter and spindle used to measure the viscosity was SC 4-18. The speed of the spindle was adjusted to 60 0.5 rpm. Data obtained were the average of five determinations and were expressed as centipoises (cp). 2.3.9. Determination of turbidity The turbidity of 6.67% gelatin solution (w/v) which was prepared with distilled water was determined using a turbidimeter (2100P HACH 46500-00, HACH Company, Colorado, US). Readings were carried out in triplicates for all samples. 2.3.10. Determination of isoelectric point The isoelectric points of the extracted gelatins were determined by running the isoelectric focusing using Bio-Rad PROTEAN IEF Cell (Bio-Rad Laboratories Inc., Richmond, CA, USA) with the isoelectrofocusing ReadyStrip IPG Strips pH range 3e10 (Bio-Rad). The runs for each gelatin were carried at least 3 times. 2.3.11. Determination of gel strength and pH The gel strength and the pH of gelatins were measured according to the method recommended by the manufacturer of the
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Table 1 Yield of gelatins from three selected freshwater fishes. Types of fish
Red tilapia Walking catfish Striped catfish a
Yielda Wet basis (%)
Dry basis (%)
Protein basis (%)
12.92 13.06 11.17
39.97 32.06 26.23
44.44 42.12 33.15
Values were means of readings from two separate batches.
texture measuring instrument. The gel strength was determined using TA-XT2 Texture Analyzer Stable Micro Systems (England) with 0.500 Radius Cylinder Probe (P/0.5R) as recommended by the manufacturer. A gelatin solution of 6.67% (w/v) was prepared with distilled water and transferred into a standard Bloom jar. The gelatins were left to mature for 17 1 h at 10 C in a water bath. After maturation period, the jars were removed immediately from the water bath and placed centrally under the probe and the penetration test was commenced. The load cell used was 5 kN. The unit for the response was recorded as grams (g). The pH was measured with a glass electrode (Toledo 320 pH Meter, Mettler-Toledo Instrument, Greifensee, Switzerland) after standardizing with pH 4.0 and 7.0 buffers. The gel strength and pH determination were carried out in triplicates. 2.3.12. Statistical analysis All data collected were analysed using Analysis of Variance (ANOVA) and Duncan’s Multiple Test to determine the significance level among the means using the statistical programme analyses package (SAS, 1989). 3. Results and discussion 3.1. Yield of gelatin The recovery of gelatin based on wet, dry and protein basis is as shown in Table 1. It was observed that the extractability of gelatin among the three species varies. The highest yield was obtained from red tilapia skin (dry weight basis). The result was higher than that reported by Jamilah and Harvinder (2002) for acid extracted gelatin but lower than those reported by Grossman and Bergman (1992) and Holzer (1996) for the same fish species. The yields of gelatin (protein basis) from walking and striped catfish skins were higher than reported by Yang et al. (2008) for channel catfish skin, which used NaOH for the alkaline pretreatment. This suggested that the use of CaOH2 to condition the skin prior to gelatin extraction resulted in better recovery. 3.2. Proximate composition of fish skins and gelatin Table 2 shows the protein, moisture and ash content of the skins of the three selected freshwater fish skins and the extracted gelatins. The skin of red tilapia skin has the highest moisture content, followed by that of walking and striped catfish. All the three freshwater fish skins had less ash contents (0.46e0.51%) compared
to that of the Nile perch skin (3.7%) (Muyonga et al., 2004). They also had higher protein contents compared to that of Nile perch reported by Muyonga et al. (2004). The maximum possible yield of gelatin to be extracted from the skin is related to the protein content of the collagenous material in the skin although it is not a direct indication. The crude protein content of the skin is therefore not a good indicator of the possible protein content of the gelatin obtained in this procedure. Of late, the composition of hydroxyproline (hyp) in the collagenous material has been suggested as a better indicator to determine the yield of gelatin extracted from fish skin (Nalinanon, Benjakul, Visessanguan, & Kishimura, 2008). However, the amino acid analytical procedure used in this experiment did not allow the detection of hydroxyproline. The ash content of all the gelatins obtained was comparatively low (0.08e0.18%). This was probably due to the ionization process that reduced the mineral content. Gelatin from skate (Raja kenojei) fish skin, which was also produced by the liming process but without ionization process, had higher ash content of approximately 1.4% (Cho, Jahncke, Chin, & Eun, 2006). 3.3. Physical characteristics of the fish gelatins All the determined physical properties of the extracted gelatins were tabulated in Table 3. Great variations in the gel strengths of the gelatins obtained were observed (Table 3). The highest strength was obtained in red tilapia gelatin (384.9 g), followed by walking and striped catfish, 238.9 g and 147.4 g, respectively. They were significantly different from each other. Gelatin obtained from red tilapia skin by liming process had higher Bloom strength, which was approximately 3 times higher than that reported by Jamilah and Harvinder (2002) for gelatin obtained through acid and alkaline conditioning process (gelatin Bloom strength was 128.1 g). Therefore, it is highly possible to produce gelatin of different gel strength from the same source by merely varying the extraction method even though the gel strength has always been associated to the natural characteristics of the protein matrix of the source. Suggestions such as varying the pH and the isoelectric point closer to the natural isoelectric point of the protein to produce stiffer and more compact gels had also been put forth (Gudmundsson & Hafsteinsson, 1997). Inducing the increase in the cross-linking of the gelatin, and thus increasing the gel strength of the gelatin with the addition of 1.0 mg/g transglutaminase has been recently reported (Norziah, Hassan, Khairulnizam, Mordi, & Norita, 2009). The gel strength of both gelatins from red tilapia and striped catfish obtained by this process were comparable to the commercial gel strengths of those from bovine and porcine origin (200e240 g). Therefore, these gelatins can be used as a substitute or complementary ingredient in required applications. Slight variations in the melting points of the gelatins were noted. However, they were higher than reported for skate (R. kenojei) fish skins which were in the range of 18e21 C (Cho et al., 2006). The melting temperature of striped catfish gelatin was similar to that of gelatin from Nile perch skin (Muyonga et al., 2004).
Table 2 The proximate compositiond of the three selected freshwater fish skins and gelatins. Fish species
Red tilapia Walking catfish Striped catfish
Moisture
Protein
Ash
Skin
Gelatin
Skin
Gelatin
Skin
Gelatin
70.43 0.26a 62.47 0.34b 60.52 0.52c
8.51 0.21a 7.86 0.53ab 7.29 0.42a
29.07 0.31a 31.01 0.48b 33.70 0.75c
93.25 0.21a 77.88 0.53b 80.02 0.33c
0.51 0.09a 0.52 0.23a 0.46 0.58a
0.18 0.01a 0.26 0.30a 0.08 0.17a
Values with the different superscripts within each column were significantly different (p < 0.05). d Values were means SD from triplicate determinations.
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Table 3 The physico-chemical propertiesd of the gelatins obtained from the three selected freshwater fish. Properties
Red tilapia
Gel strength (g) Melting point ( C) Viscosity (cp) Turbidity (NTU) Isoelectric point pH at 25 C Hunter colour value ‘L’ ‘a’ ‘b’ Sensory attributes Colour appearance (powder)
384.9 27.77 7.70 176 4.2 5.38
Whitish Shiny and soft textured
Odour (powder) Odour after redissolving
Barely detectable fishy odour Slightly fishy when re-dissolved
Walking catfish
4.49a 0.25a 0.23a 6.5a 0.5a 0.02a
147.40 24.96 6.28 495 5.3 5.02
79.45 1.10a 0.71 0.09a 5.75 0.14a
12.29b 0.25b 0.83b 6.5b 0.4b 0.08b
67.37 0.53b 2.37 0.04b 7.48 0.50b Dark brownish Shiny and slightly coarse texture Emitted slightly fishy odour Detectable fishy odour
Striped catfish 238.90 26.20 8.21 511 5.1 5.12
8.54c 0.26c 0.57c 13.1c 0.2b 0.03b
68.69 0.10c 2.66 0.04c 7.98 0.24c Light brownish Shiny and medium soft texture Detectable fishy odour Detectable fishy odour
Values with the different superscripts within each row were significantly different (p < 0.05). d Values were means SD from triplicates determination.
Viscosity is the second most important commercial physical property of a gelatin (Wards & Courts, 1997), which is partially controlled by molecular weight and polydipersity of the peptides. Higher molecular weight will increase viscosity but the polydipersity can have various effects depending on the molecular weight distribution (Gudmundsson & Hafsteinsson, 1997). The viscosities of the gelatins obtained were significantly different (p < 0.05). Their viscosities values were in the range of 6e9 cp. Grossman and Bergman (1992) reported tilapia skin gelatin had a viscosity of 5.1 cp. Lower viscosity (<3.0 cp) was reported for gelatin from channel catfish (Yang et al., 2008). The above results thus indicate that natural variations in the viscosity can be expected from different freshwater fish species, although the extraction methods do play a role. Turbidity or degree of clarity of a solution is very important in food applications such as in the addition of gelatin as a thickening agent. Turbidity values (Table 3) of the gelatins extracted from the three fish species were in the range of 176e511 NTU. Red tilapia skin gelatin exhibited lower turbidity than the gelatins obtained from either striped or walking catfish. Muyonga et al. (2004) stated that efficiency of the filtration process during gelatin extraction affected the degree of turbidity of gelatin solution, which may explain the results obtained for striped and walking catfish gelatins. Table 4 Amino acids contents (mg/g) of the gelatins obtained from the three selected freshwater fishes. Amino acid (mg/g)a
Red tilapia
Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys Total
43.94 71.29 31.60 197.00 ND ND 42.11 20.07 123.71 5.23 15.74 15.20 ND ND 11.88 34.32 8.04 620
1.05 1.15 0.11 0.21
0.98 1.24 2.73 0.23 0.39 1.44
0.28 1.44 1.75
Walking catfish
Striped catfish
55.44 79.27 35.95 204.13 ND 75.95 21.95 88.41 122.65 7.63 21.60 20.86 ND 11.72 23.47 27.94 35.15 832
46.79 78.94 30.79 209.80 ND 79.39 36.23 96.20 136.13 6.45 23.97 22.03 ND 16.24 36.35 26.21 25.75 871
2.68 4.58 4.37 15.68 3.80 3.21 2.27 9.94 0.64 2.40 1.99 1.51 1.53 1.62 1.97
ND e not detected. a Values are presented as the mean SD of three readings.
5.42 3.71 0.70 9.32
11.35 12.26 9.48 5.31 1.11 2.44 1.47
1.54 4.67 3.84 11.02
The turbidity value of red tilapia skin gelatin was closed to that obtained for Nile perch skin gelatin extracted at 60 C (Muyonga et al., 2004). The isoelectric points of the fish gelatins were in the range of 4.2e5.3, which were typical for type B gelatin. No earlier reports could be found to compare our results. Colour characteristics were also different among the gelatins and this could be due to the effect of the carry over of pigments from their skins. The ‘L’ (lightness) values of the three gelatins are significantly different (p < 0.05) and this confirms the visual observations on the gelatins. The colour of the red tilapia gelatin obtained in this study was different from that reported by Jamilah and Harvinder (2002) for the same fish species. The reported ‘L’ value for red tilapia gelatin using acidic treatment was lighter (92.4). Fish odour in the red tilapia gelatin was barely detectable but in both of the catfish species, fishy odour was slightly stronger. The odour description of red tilapia gelatin is similar to that of Jamilah and Harvinder (2002). They suggested that stronger fishy odour is correlated to the muddy odour and flavour associated with the fish species and also perhaps related to the feed of the fish. 3.4. Amino acids composition The total amino acid concentration was highest in striped catfish, followed by walking catfish and red tilapia. The rigidity of the gelatin gel is directly related to its proline (Pro) and hydroxyproline (Hyp) contents (Holzer, 1996). In this study, Hyp was not determined due to the inability of method employed to detect its presence as mentioned earlier. Glycine and proline, were the two main amino acids which made up a quarter (w25%) of the total amino acids content (Table 4). The highest glycine and proline contents were found in the striped catfish. The composition of amino acids for sole, megrim, cod, hake and squid was reported to be similar to the composition of interstitial collagen, accounting to >30% Gly and w17% imino acids (Pro þ Hyp) (Gómez-Guillén et al., 2002). A higher amino acid content of Pro, Hyp and Ala in commercial gelatin from tilapia has been previously shown as one of the major cause for its higher viscoelastic properties than megrim gelatin (Gómez-Guillén, Sarabia, & Montero, 2000). Glutamine as the third highest amino acid in all the extracted gelatin and no obvious difference between that of walking catfish and the striped catfish. Alanine was significantly higher in walking catfish and striped catfish as compared to the red tilapia. The values were approximately 4e5 times higher to that found in the red tilapia. Both isoleucine (Ile) and arginine (Arg) were not detected in
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the tilapia gelatin. Cystine (Cys) and histidine (His) were also not detected in all extracted gelatins. 4. Conclusion This study has proven that the liming process employed is a viable process for the extraction of the gelatin from fish skins to produce higher Bloom. The characteristics of the gelatins obtained in this process indicate that they have good commercial application. The red tilapia gelatin showed superior characteristics as compared to the other two species studied. Acknowledgement The authors would like to extend their sincere thanks to the Malaysian Ministry of Science and Technology (MOSTI) for providing the Intensive Research Priority Area (IRPA) grant to carry out this study. References AOAC. (1995). Official method of analysis. Washington D.C., USA: Association of Official Chemist. Arnesen, J. A., & Gildberg, A. (2007). Extraction and characterisation of gelatine from Atlantic salmon (Salmo salar) skin. Bioresource Technology, 98, 53e57. Choi, S. S., & Regenstein, J. M. (2000). Physico-chemical and sensory characteristic of fish gelatin. Journal of Food Science, 65, 194e199. Cho, S. H., Jahncke, M. L., Chin, K. B., & Eun, J. B. (2006). The effect of processing conditions on the properties of gelatin from skate (Raja kenojei) skins. Food Hydrocolloids, 20, 810e816. GME Market data. (2007). Official website of GME. Brussels, Belgium: Gelatin Manufacturers of Europe. http://www.gelatine.org GME Market Data. Gómez-Guillén, M. C., & Montero, P. (2001). Extraction of gelatin from megrim (Lepidorhombus boscii) skins with several organic acids. Journal of Food Science, 66(2), 213e216. Gómez-Guillén, M. C., Sarabia, A. I., & Montero, P. (2000). The effect of added salts on the viscoelastic properties of fish skin gelatins. Food Chemistry, 70, 71e76. Gómez-Guillén, M. C., Turnay, J., Fernandez-Diaz, 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 Hydrocolloids, 16, 25e34. Grossman, S., Bergman, M. (1992). Process for the production of gelatin from fish skins. U.S. patent 5,093,474.
Gudmundsson, M., & Hafsteinsson, H. (1997). Gelatin from cod skins as affected by chemical treatments. Journal of Food Science, 62, 37e47. Holzer, D. (1996). Gelatin production. U.S. patent 5,484,888. Irwandi, J., Faridayanti, S., Mohamed, E. S. M., Hamzah, M. S., Torla, H. H., & Che Man, Y. B. (2009). Extraction and characterisation of gelatin from different marine fish species in Malaysia. International Food Research Journal, 16, 381e389. Jamilah, B., & Harvinder, K. G. (2002). Properties of gelatins from skins of fish-black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica. Food Chemistry, 77, 81e84. Karim, A. A., & Bhat, R. (2009). Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocolloids, 23, 563e576. Kittiphattanabawon, P., Benjakul, S., Visessanguan, W., Nagai, T., & Tanaka, M. (2005). Characterisation of acid-soluble collagen from skin and bone of bigeye snapper (Priacanthus tayenus). Food Chemistry, 89, 363e372. Kolodziejska, I., Kaczorowski, K., Piotrowska, B., & Sadowska, M. (2004). Modification of the properties of gelatin from skins of Baltic cod (Gadus morhua) with transglutaminase. Food Chemistry, 86, 203e209. Kolodziejska, I., Skierka, E., Sadowska, M., Kolodziejski, W., & Niecikowska, C. (2008). Effect of extracting time and temperature on yield of gelatin from different fish offal. Food Chemistry, 107, 700e706. Muyonga, J. H., Cole, C. G. B., & Duodu, K. G. (2004). Extraction and physico-chemical characterisation of Nile perch (Lates niloticus) skin and bone gelatin. Food Hydrocolloids, 18, 581e592. Nalinanon, S., Benjakul, S., Visessanguan, W., & Kishimura, H. (2008). Improvement of gelatin extraction from bigeye snapper skin using pepsin-aided process in combination with protease inhibitor. Food Hydrocolloids, 22, 615e622. Norziah, M. H., Al-Hassan, A., Khairulnizam, A. B., Mordi, M. N., & Norita, M. (2009). Characterization of fish gelatin from surimi processing wastes: thermal analysis and effect of transglutaminase on gel properties. Food Hydrocolloids, 23, 1610e1616. Ockermann, H. W., & Hansen, C. L. (1988). Glue and gelatin. In Animal by-product processing (pp. 132e157). England, Chichester: Elli Horwood Ltd. SAS. (1989). Statistical analysis system for data analysis. North Carolina, USA: The SAS Institute. Schrieber, R., & Gareis, H. (2007). Manufacture of gelatin: theory and practice. In Gelatin handbook, theory and industrial practice (pp. 42e88). Weinheim: Wiley-VCH. Stanley, F. D. (2002). Improved alkaline process for preparing type B fish gelatin. World Intellectual Property Organization. WO/2002/094959. Ward, A. G., & Courts, A. (1977). The science and technology of gelatin. London: Academic Press. Yang, H., Wang, Y., Zhou, P., & Regenstein, J. M. (2008). Effects of alkaline and acid pretreatment on the physical properties and nanostructures of the gelatin from channel catfish skins. Food Hydrocolloids, 22, 1541e1550. Zhou, P., & Regenstein, J. M. (2005). Effects of alkaline and acid pretreatments on Alaska pollock skin gelatin extraction. Journal of Food Science, 70(6), C392eC396.