BLBS102-c20 BLBS102-Simpson March 21, 2012 13:35 Trim: 276mm X 219mm Printer Name: Yet to Come
20 Fish Collagen 373Nalinanon et al. (2007) reported that the addition of BSP at a
level of 20 kUnits/g of defatted skin resulted in an increased
content of collagen extracted from bigeye snapper skin. The
yields of collagen from bigeye snapper skin extracted for 48
hours with acid and with BSP were 5.31% and 18.74% (dry
basis), respectively. With preswelling in acid for 24 hours,
collagen extracted with BSP at a level of 20 kUnits/g of defatted
skin for 48 hours had a yield of 19.79%, which was greater than
that of collagen extracted using porcine pepsin at the same level
(13.03%). When tuna pepsin (10 units/g defatted skin) was used
for collagen extraction from the skin of threadfin bream for
12 hours, the yield of collagen increased by 1.84- to 2.32-fold
and albacore pepsin showed the comparable extraction efficacy
to porcine pepsin (Nalinanon et al. 2008). Recently, PSCs from
the skin of two species of bigeye snapper,P. tayenusandPri-
acanthus macracanthus, were extracted with the aid of tongol
tuna (Thunnus tonggol) pepsin and porcine pepsin (Benjakul
et al. 2010). Yields of PSCs extracted from the skin ofP. tayenus
with the aid of porcine pepsin (T-PP) and tongol tuna pepsin
(T-TP) were 77.4 and 87.3 g kg−^1 (based on wet weight of skin),
respectively, while those of PSCs extracted from the skin ofP.
macracanthuswith the aid of porcine pepsin (M-PP) and tongol
tuna pepsin (M-TP) were 70.6 and 72.9 g kg−^1 , respectively. For
the same pepsin used,P. tayenusskin gave a higher yield thanP.
macracanthusskin. When the same skin was used for collagen
extraction, tongol tuna pepsin treatment resulted in the higher
yield (Benjakul et al. 2010). Therefore, tuna pepsin showed
higher efficiency in collagen extraction. Pepsin from different
sources might have a different cleavage site on the collagen.
As a result, the peptides in the telopeptide region, which is
susceptible to hydrolysis, were hydrolyzed to different degrees
(Nalinanon et al. 2008, Benjakul et al. 2010). In addition, col-
lagen from different sources was probably cleaved by the same
pepsin in different ways (Benjakul et al. 2010). Furthermore,
collagen from the two skins may have some differences in
configuration and amino acid sequence. Fish pepsins used for
collagen extraction from several sources exhibited the compa-
rable extraction efficacy to porcine counterpart. Therefore, fish
pepsin could be used as a replacer of mammalian pepsin.
The extraction conditions for ASC and PSC from different
sources and their yield, type, and molecular compositions are
summarized in Table 20.4.Recovery of CollagenAfter extraction, the collagen is generally recovered by salt
precipitation prior to dialysis and freeze-drying. The colla-
gen solution is generally precipitated by adding NaCl to a
final concentration of 2.6 M in the presence of 0.05 M
tris(hydroxylmethyl)aminomethane, pH 7.5 (Nagai and Suzuki
2000, Jongjareonrak et al. 2005, Kittiphattanabawon et al. 2005,
Nalinanon et al. 2007, Benjakul et al. 2010). However, the con-
centrations of NaCl used for collagen precipitation in different
methods are varied from 0.9 to 2.6 M. This can be adjusted
to maximize the collagen recovery and removal of impurities.
The resultant precipitate is then collected by centrifugation. The
pellet is dissolved in 0.5 M acetic acid with minimal volumeprior to dialysis against 0.1 M acetic acid and distilled water.
The dialysate is finally freeze-dried and the powder obtain is
used as collagen. Collagen can be isolated from fish skin, bone,
and scale (Nomura et al. 1996, Nagai et al. 2002a, Ikoma et al.
2003, Mizuta et al. 2003, Muyonga et al. 2004, Ogawa et al.
2004, Nalinanon et al. 2007, Nalinanon et al. 2008). Nagai and
Suzuki (2000) extracted type I collagen from underutilized re-
sources including fish skin, bone, and fin with varying yields
as follows: (1) skin collagen: 51.4% (Japanese sea bass), 49.8%
(chub mackerel), and 50.1% (bullhead shark); (2) bone colla-
gen: 42.3% (skipjack tuna), 40.7% (Japanese sea bass), 53.6%
(ayu), 40.1% (yellow sea bream), and 43.5% (horse mackerel);
(3) fin collagen: 5.2% (Japanese sea bass acid-soluble colla-
gen) and 36.4% (Japanese sea bass acid-insoluble collagen) on
the dry basis. Kittiphattanabawon et al. (2005) extracted ASC
from the skin and bone of bigeye snapper (P. tayenus) with
the yields of 10.94% and 1.59% on the basis of wet weight,
respectively. The yield of ASC from the skins of young and
adult Nile perch (Lates niloticus) extracted using 0.5 M acetic
acid and precipitation with 0.9 M NaCl were 63.1% and 58.7%
(on a dry weight basis), respectively (Muyonga et al. 2004).
Sadowska et al. (2003) extracted the collagen from the skins of
Baltic cod (G. morhua). The use of a one-stage, 24-hour extrac-
tion of whole skins with acetic at ratio of material to solvent
of 1:6, yielded 20% collagen. Using three consecutive 24-hour
extractions of whole skins with citric acid, 85% of collagen pro-
tein could be separated. Recently, ASC and PSC from the skin
of brownbanded bamboo shark (Chiloscyllium punctatum) were
isolated and characterized by Kittiphattanabawon et al. (2010a).
The yield of ASC and PSC were 9.38% and 8.86% (wet weight
basis), respectively. Collagen from the outer skin of cuttlefish
(Sepia lycidas) was extracted by Nagai et al. (2001). The initial
extraction of the cuttlefish outer skin in acetic acid yielded only
2% of collagen (dry weight basis). On subsequent digestion
of the residue with 10% pepsin (w/v), a solubilized collagen
(PSC) was obtained with a yield of 35% (dry weight basis).
Nagai et al. (2000) found that 35.2% collagen was extracted
from rhizostomous jellyfish (Rhopilema asamushi) by limited
pepsin digestion.CHARACTERISTICS AND PROPERTIES
OF COLLAGENSCollagen from different sources generally has the varying prop-
erties, especially the susceptibility to thermal denaturation or
cleavage by proteases (Nalinanon et al. 2007, Nalinanon et al.
2010). Fish collagen, comprising lower imino acid content, is
less stable than mammalian collagen. This property directly af-
fected the application.Mammalian CollagenBovine skin corium obtained from freshly slaughtered cattle
of increasing biological age showed greater resistance to the
action of acids, as measured by extractable collagen (Miller
et al. 1983). Citrate-soluble content of corium from fetal skin
was 30.9%, while that of 3–6-week-old calf was 6.4% and of