Fish Gelatin

Abstract

Gelatin is obtained by the hydrolysis of collagen which is the principal protein found in skin and bones. Fish gelatin is being extracted commercially in Nova Scotia by Kenney & Ross at Port Saxon. The raw material is the skin from deep water fish such as cod, haddock and pollock, and is obtained from local salt fish and frozen fish processors. The uniqueness of fish gelatin lies in the amino acid content of the gelatin. Although all gelatins are composed of the same 20 amino acids, there can be a variation in the amount of imino acids, proline and hydroxyproline. With lower amounts of these imino acids, there is less hydrogen bonding of gelatin in water solutions, and hence a reduction in the gelling temperature. Gelatin from cod skin gels at 10ºC, whereas gelatin from carp skin would be more similar to animal gelatin, which gels above room temperature. Most people think of gelatin as a food additive or part of photographic film. With a lower gelling temperature, other commercial applications of fish gelatin have been developed. These applications are discussed.

Overview

The amount of information reported in the literature on fish gelatin is somewhat limited (Mees, 1966; Gustavson, 1956; Piez, 1965). Kenney & Ross has been producing fish gelatin since 1960, and this conference gives us the opportunity to share with you some of the information that we have developed. The product initially manufactured by us was marketed as a clarified fish glue, and essentially was a fish gelatin used in industrial application. We have been marketing an edible type of fish gelatin since 1981.

All gelatin is derived from collagen, the principal protein found in skin and bone. A simplified characterization of the applications of gelatin would be into the following four uses:

1. Edible gelatin - Free of heavy metals and aesthetically suitable for eating.
2. Industrial gelatin - Where the chemical and physical properties are uniquely suitable for an industrial application. A good example would be gelatin used for the microencapsulation of dye precursors for carbonless paper.
3. Photographic gelatin - The requirements being extremely critical. Photographic film requires a long shelf-life, and the gelatin has a major impact on the silver halide chemistry that requires the ability to take a picture and be able to develop it later with standard developing conditions.
4. Glue - Essentially for adhesive or gluing applications.

Fish gelatin, with the exception of photographic film, is used in all these applications. However, fish gelatin is used as the base for a light sensitive coating (or photoresist) for the electronics trade.

The precursor for gelatin is collagen. Collagen is the major structural protein found in the skin and bones of all animals. The collagen molecule consists of 3 individual polypeptide chains (alpha chains) which are wound around one another in a triple helix confirmation. This triple helix is stabilized by hydrogen bonds between collagen molecules, which happens as the animal ages. A collagen molecule of three alpha chains would measure 3000Aº in length (0.3 microns) and 15Aº in diameter. Each alpha chain has approximately 1050 amino acids connected together. There are twenty different amino acids in each alpha chain, and for each animal type of gelatin, these amino acids are in a specific repeated pattern. Glycine, which represents a third of the amino acids content, is in repeated sequence with two other amino acids. This might be represented as glycine-x-y. It is not unusual for x to be proline and y to be a hydroxyproline residue.

Table 1 compares the content of amino acids in fish gelatin with that of calf skin gelatin. We sell our gelatin as HiPure Liquid and Dry Gelatin. The amino acids are listed alphabetically together with their functionality. A common way of stating the content of amino acids is by indicating the number of residues per thousand amino acids. Note the comparison between fish gelatin and calf skin gelatin for the amino acids proline and hydroxyproline.

Table 1 Comparative contents of amino acids in Hi-Pure liquid gelatin and animal gelatin.
Amino Acid	Function	Residues/1000 Amino Acids
		HiPure Liquid Gelatin	Calf Skin Gelatin
Alanine Arginine Aspartic Acid	Non polar aliphatic Basic amino acid Dicarboxylic acid	124 55 37	114 51 45
Cysteine Glutamic Acid Glycine	Sulphur containing Dicarboxylic acid Non polar aliphatic	-- 77 334	-- 71 325
Histidine Hydroxyproline Isoleucine	Basic amino acid Imino acid Non polar aliphatic	9 54 10	5 86 11
Leucine Lysine Methionine	Non polar aliphatic Basic amino acid Sulphur containing	20 35 13	25 34 6
Phenylalanine Proline Serine	Aromatic Imino Acid Hydroxyl containing	11 106 65	13 135 37
Threonine Tryptophan Tyrosine	Hydroxyl containing Aromatic Hydroxyl containing	26 -- 2	18 -- 3
Valine	Non polar aliphatic	22	22

The role of proline and hydroxyproline in collagen is very critical. There appears to be a relationship between the temperature at which the animal (or fish) metabolizes and the properties of the skin and resultant extracted gelatin. Gelatin derived from the skin of deep cold water fish has lower amounts of proline and hydroxyproline, and as a result, water solutions will not gel at room temperature, but will remain liquid to 8 to 10ºC.

Collagen in skin can be dissolved in dilute cold acid or salt solution, but with great difficulty and generally poor yields. This is because collagen molecules are covalently cross-linked into fibrils that may swell, but do not dissolve. If the skin and cold acid are heated, at a certain temperature the collagen molecule undergoes a helix coil transition. The helix coil literally unfolds, and the collagen becomes more readily soluble. The temperature at which this occurs depends upon the amount of proline and hydroxyproline in the alpha chain, and this temperature is the point of denaturing. For deep cold water fish collagen, this temperature is approximately 15ºC. Bovine collagen is approximately 40ºC. Another term, which covers the same effect in the skin itself, is the shrinkage temperature. This is exactly what the term implies. At a certain temperature, the collagen in the raw skin will relax and the skin will shrink. The shrinkage temperature of the raw skin is higher than the denaturing temperature of the skin in acidified water, but the same relationship of proline and hydroxyproline to the shrinkage temperature still holds.

Table 2 shows a comparison of the amino acid composition of some fish collagens that have been published by Piez and Gross (1960). Glycine represents a third of the total amino acids residues. There are similarities in the composition, but what is important, is the total amount of proline and hydroxyproline, as well as the highest shrinkage temperature.

Table 2 Amino acid compositions and shrinkage temperatures of fish and calf gelatins.*
Amino Acid/ Shrinkage	Residues/1000 Amino Acids
	Carp Skin Gelatin	Cod Skin Gelatin	Pike Skin Gelatin	Calf Skin Gelatin
Glycine Alanine Valine	317 120 19	345 107 19	328 114 18	320 112 20
Isoleucine Leucine Proline	12 25 124	11 23 102	9.2 20 129	11 25 138
Hydroxyproline Phenylalanine Tyrosine	73 14 3.2	53 13 3.5	70 14 1.8	94 13 2.6
Serine Threonine Methionine	43 27 12	69 25 13	41 25 12	36 18 4.3
Cystine Hydroxylysine Lysine	<1 4.5 27	<1 6.0 25	<1 7.9 22	<1 7.4 27
Histidine Arginine Aspartic Acid	4.5 53 47	7.5 51 52	7.4 45 54	5.0 50 45
Glutamic Acid	74	75	81	72
Shrinkage Temp (°C)	57	40	55	65

*Piez and Gross (1960)

Commercial extraction of gelatin depends upon both dissolving and hydrolyzing the denatured skin. The gelatin may retain some covalent bonds between alpha chains, which would entail multiples of the single alpha chain length of 95,000 daltons. There are also major proportions of shorter chain polypeptide in the gelatin, as the chain is cleaved in the extraction process. This is not necessarily a problem, as the end product may not need very high molecular fractions in order to accomplish the specific application.

The molecular weight of four gelatins were compared using gel permeation chromatography and low angle light scattering (Berg and Frederick, private communication) in Fig. 1. The top curve is a standard gelatin, and underneath it is calf skin gelatin. The second curve is fish gelatin extracted with minimal hydrolysis. The lowest curve is commercial fish gelatin, which was hydrolyzed more to achieve certain viscosity characteristics. The animal gelatins and the low hydrolysis fish gelatin all have peaks at 190,000 and 95,000, characteristic of beta and alpha collagens respectively. About 10% of the wet weight of fish skin is Type I collagen. Fig. 2 is a comparison of molecular composition of calf skin collagen with fish skin collagen, done by using polyacrylamide gel electrophoresis (PAGE) according to the method of Laemml (1970), whereby molecules of different molecular weights are separated using an electrical current. There are similarities between the two collagens. The fish collagen consisted primarily of alpha 1 and alpha 2 chains in a 2:1 ratio. Beta collagen would be two alpha collagens covalently linked together.

Fig. 3 plots the viscosity of a 10% solution of animal gelatin vs fish gelatin with minimal hydrolysis and commercial fish gelatin. There are similarities between the gelatins, but note that the animal gelatin gels at 32ºC, whereas the fish gelatin remains liquid down to 8ºC.

Norland HiPure Liquid Gelatin is supplied at 45% solids in water and is pourable solution at 7000 to 8000cps, about the viscosity of honey. The gelatin is protected by a mixture of methyl and propyl hydroxybenzoates.

Fish gelatin has similar chemical reactivity to animal gelatin. Aldehydes such as formaldehyde, gluteraldehyde and glyoxal will cross-link and harden the gelatin under appropriate conditions. It can be reacted with anydrides under alkaline conditions, reducing or eliminating the effect of aldehydes as a hardening agent on the gelatin. Fish gelatin also provides a good medium for precipitating silver halide emulsions, as this can be done at lower temperatures than with animal gelatin.

Our factory is located on the southwestern tip of Nova Scotia, Canada, in an area rich in fishing grounds. Skins are obtained from frozen fish producers, as well as salt fish processors. Large quantities of skin are used in the plant, and 16,000 pounds of skins are handled daily.

I mentioned fish gelatin being used in a light sensitive coating. Our clarified fish glue was originally used as a base for a water soluble photoresist (Holahan, 1965). If you have a color television set there is a good possibility that the critical part of the television tube, the aperture mask, was made using a photolithographic process with fish gelatin as the photoresist base. There are 400,000 holes or slots in the mask and the purpose of these holes is to delineate the color picture that is projected onto the phosphors on the inside of the television tube. The optics are so critical, that imperfections of a fraction of a micron can be visually seen on the screen, and would be a cause for rejection. These masks are made in a continuous process whereby thin metal is unwound from a role from one end of the equipment, and completed masks are stripped from the sheet at the other end 1,000 feet away.

Fish gelatin is also used in the manufacture of lead frames that hold the silicon chip in computers and microprocessors (American Machinist, 1971). The chip is mounted on a pad at the center portion of the lead frame, and each circuit on the chip is connected to a lead that surrounds it. The lead frame are made using a photochemical machining process and a fish gelatin photoresist.

A necessary part of the optics of a color video camera is a color stripped filter that separates the color signals for the electronics of the camera. Each filter has a series of 3 different color stripes, 12 microns wide, put down on glass in a repeat pattern. The total size of the filter is approximately 1/2" x 3/4". The stripes are put on the glass using a photolithographic process in the fish gelatin.

Summary

1) Gelatin is made from collagen, which is part of the skin and bones of animals and fish.

2) All gelatins have same 20 different amino acids, in slightly different proportions for different species.

3) The amount of the imino amino acids, proline and hydroxyproline, determines the shrinkage temperature and the denaturing temperature, (The temperature at which the collagen helix unwinds), and as a result, the temperature at which solutions of the extracted gelatins gels.

4) Gelatin from cold deep water fish such as cod, haddock, pollock, hake and cusk, gels at 8 to 10ºC compared to calf skin gelatin which gels at 30 to 35ºC.

5) Fish gelatin is used in a variety of coating applications, the largest of which is a base for a water soluble photoresist.

References

Berg, R. and Frederick. Private communication. Biomaterials Centre, Dept. of Pathology, Rutgers Medical School, Piscataway, NJ.

Burjanadze, T.V. 1982. Stabilization of collage structure; Dependence of collagen denaturation enthalpy on the imino acid content. Biopolymers 21(8): 1587.

Chemically Milling Precision Parts. 1971. American Machinist 115: 50.

Gustavson, K.H. 1956. "The Chemistry and Reactivity of Collagen." Academic Press, New York, NY.

Holahan, J.F. 1965. Manufacture of color picture tubes. Electronics World 74(6):30-32,56.

Laemmll, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259): 680.

Mees, C.E.K. and James, T.H. 1966. "The Theory of the Photographic Process." 3rd ed. Macmillan Company, New York.

Piez, K. 1965. Characterization of a collagen from cod skin containing three chromatographically different chains. Biochemistry 4(12): 2590.

Piez, K.A. and Gross, J. 1960. The amino acid composition of some fish collagens: The relations between composition and structure. J. Biol. Chem. 235(4): 995.

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