325
2005 ), bigeye snapper (Priacanthus tayenus) (Kittiphattanabawon et al. 2005 ), ocellate puffer fi sh ( Takifugu rubripes ) (Nagai et al. 2002 ), redfi sh ( Sebastes mentella )
(Wang et al. 2007b ), walleye pollack ( Theragra chalcogramma ) (Yan et al. 2008 ),
yellowfi n tuna ( Thunnus albacares ) (Woo et al. 2008 ), hoki ( Macruronus novaezelandiae ) and ling ( Genypterus blacodes ) (Hofman and Newberry 2011 ).
Methods of collagen isolation from fi sh skin differ in details. However, there are
some steps which are common for all methods used. Here, as example, I give briefl y
description of isolation of collagen from skin of brown backed toadfi sh ( Lagocephalus
gloveri ) according to Senaratne et al ( 2006 ):
“All the preparative steps were carried out at 4 °C with continuous stirring. Skin
pieces were treated with 0.1 N NaOH to remove non-collagenous proteins at a solid
to solution ratio of 1:10 (w/v) for 3 days. After this, they were washed with distilled
water till the washed water became neutral or slightly basic pH. The alkali solution
was changed every day. Then, the sample was defatted with 10 % butyl alcohol at a
solid to solvent ratio of 1:10 (w/v) for 24 h, washed with ample amounts of distilled
water, and lyophilized. The lyophilized matter was extracted with 0.5 M acetic acid
for 3 days. Then, 10 % of pepsin (1:10,000 units) according to the lyophilized
weight was added and hydrolysed for 48 h. The viscous solution was centrifuged at
12,000 g for 1 h at 4 °C and supernatant was collected. The supernatant was salted
out by adding NaCl to a fi nal concentration of 0.7 M. This was followed by precipitation by addition of NaCl to a fi nal concentration of 2.3 M in 0.05 M Tris–HCl
(pH 7.5). The resultant precipitate was separated by centrifugation at 12,000 g for
1 h at 4 °C. The precipitate was then dissolved in 0.5 M acetic acid, dialysed against
0.1 M acetic acid, distilled water, and lyophilized,” (Senaratne et al. 2006 ).
From industrial point of view collagen isolated from the skins of freshwater
aquaculture cultivated fi sh species like carp, tilapia, and rainbow trout (Tabarestani
et al. 2012 ) are of great interest.
Fish Scale Collagen The collagen fi brils-containing basal plate of Amiidae ( Amia )
and Sarcopterygii ( Leptdosiren , Latimeria , Protopterus , Neoceratodus ) looks like
plywood, “a system of superimposed layers of parallel fi bers or fi brils the directions
of which rotate with a regular angle in two successive layers,” (Meunie 1984 ; see
also Bigi et al. 2001 ). As characterized by Meunier ( 1984 ), “the double twisted
plywood is constituted of two imbricate systems, the odd and the even, where the
rotation of the fi brillar directions is right-handed in Sarcopterygii and left handed in
Amiidae and numerous primitive Teleostei. The orthogonal plywood, with its two
main orthogonal fi brillar directions, characterizes the evolved Teleostei and some
more primitive ones,” (Meunie 1984 ). Thus, “the basal plate is a stacking of collagenous fi bred layers. In one layer, fi brils are parallel to each other. The diameter
of fi brils (30–190 nm) is greater than in bone (20–50 nm) or osseous layer (20–
30 nm),” (Meunie 1984 ; see also Meunier 1983 ; Zylberberg et al. 1988 ). Recently
Youn and Shin ( 2009 ) studied the supramolecular organization of collagen fi brils in
fi sh scales of red seabream ( Pagrus major ). These authors reported, “a collagen
fi ber consists of helical substructures of collagen fi brils wrapped with incrustation.
[…] Freshly growing edge region of fi sh scale, embedded into fi sh skin, showed
8.1 Isolation and Properties of Fish Collagens
2005 ), bigeye snapper (Priacanthus tayenus) (Kittiphattanabawon et al. 2005 ), ocellate puffer fi sh ( Takifugu rubripes ) (Nagai et al. 2002 ), redfi sh ( Sebastes mentella )
(Wang et al. 2007b ), walleye pollack ( Theragra chalcogramma ) (Yan et al. 2008 ),
yellowfi n tuna ( Thunnus albacares ) (Woo et al. 2008 ), hoki ( Macruronus novaezelandiae ) and ling ( Genypterus blacodes ) (Hofman and Newberry 2011 ).
Methods of collagen isolation from fi sh skin differ in details. However, there are
some steps which are common for all methods used. Here, as example, I give briefl y
description of isolation of collagen from skin of brown backed toadfi sh ( Lagocephalus
gloveri ) according to Senaratne et al ( 2006 ):
“All the preparative steps were carried out at 4 °C with continuous stirring. Skin
pieces were treated with 0.1 N NaOH to remove non-collagenous proteins at a solid
to solution ratio of 1:10 (w/v) for 3 days. After this, they were washed with distilled
water till the washed water became neutral or slightly basic pH. The alkali solution
was changed every day. Then, the sample was defatted with 10 % butyl alcohol at a
solid to solvent ratio of 1:10 (w/v) for 24 h, washed with ample amounts of distilled
water, and lyophilized. The lyophilized matter was extracted with 0.5 M acetic acid
for 3 days. Then, 10 % of pepsin (1:10,000 units) according to the lyophilized
weight was added and hydrolysed for 48 h. The viscous solution was centrifuged at
12,000 g for 1 h at 4 °C and supernatant was collected. The supernatant was salted
out by adding NaCl to a fi nal concentration of 0.7 M. This was followed by precipitation by addition of NaCl to a fi nal concentration of 2.3 M in 0.05 M Tris–HCl
(pH 7.5). The resultant precipitate was separated by centrifugation at 12,000 g for
1 h at 4 °C. The precipitate was then dissolved in 0.5 M acetic acid, dialysed against
0.1 M acetic acid, distilled water, and lyophilized,” (Senaratne et al. 2006 ).
From industrial point of view collagen isolated from the skins of freshwater
aquaculture cultivated fi sh species like carp, tilapia, and rainbow trout (Tabarestani
et al. 2012 ) are of great interest.
Fish Scale Collagen The collagen fi brils-containing basal plate of Amiidae ( Amia )
and Sarcopterygii ( Leptdosiren , Latimeria , Protopterus , Neoceratodus ) looks like
plywood, “a system of superimposed layers of parallel fi bers or fi brils the directions
of which rotate with a regular angle in two successive layers,” (Meunie 1984 ; see
also Bigi et al. 2001 ). As characterized by Meunier ( 1984 ), “the double twisted
plywood is constituted of two imbricate systems, the odd and the even, where the
rotation of the fi brillar directions is right-handed in Sarcopterygii and left handed in
Amiidae and numerous primitive Teleostei. The orthogonal plywood, with its two
main orthogonal fi brillar directions, characterizes the evolved Teleostei and some
more primitive ones,” (Meunie 1984 ). Thus, “the basal plate is a stacking of collagenous fi bred layers. In one layer, fi brils are parallel to each other. The diameter
of fi brils (30–190 nm) is greater than in bone (20–50 nm) or osseous layer (20–
30 nm),” (Meunie 1984 ; see also Meunier 1983 ; Zylberberg et al. 1988 ). Recently
Youn and Shin ( 2009 ) studied the supramolecular organization of collagen fi brils in
fi sh scales of red seabream ( Pagrus major ). These authors reported, “a collagen
fi ber consists of helical substructures of collagen fi brils wrapped with incrustation.
[…] Freshly growing edge region of fi sh scale, embedded into fi sh skin, showed
8.1 Isolation and Properties of Fish Collagens