268
12 Collagen
has been washed off and the system is neutral. The type of water used depends on the
level of purity required. Clean tap water could be used, or distilled water or ultrapure
water.
For the acid-based extraction, the fish waste was treated with acid in order to
dissolve out the acid-soluble collagen from the rest of the tissue. The first step was to
remove the fat using 10% butyl alcohol at a 1:10 ratio of solid to alcohol for a duration
of 8 h. The fats are alcohol soluble so it will be removed from the fish waste. The fish
waste is then washed with cold water to remove any residue of alcohol and fat from the
surface. The next stage is then to extract the collagen in acetic acid at a concentration
of 0.5 M and 1:15 solid to liquid ratio. The extraction is allowed a duration of 24 h. The
extraction time can be varied for different samples since the composition of collagen
and other components varies for different types of fish. Defatting prior to extraction
prevents the fat from being released alongside the collagen during the extraction. The
fat will dissolve in alcohol but collagen will not, however the fat could be released
into the acetic acid solution hence the order is also important. The collagen dissolved
in acetic acid is then precipitated out in a 0.05 M tris (hydroxymethyl) aminomethane
containing sodium chloride at a concentration of 2.6 M. To separate the precipitated
collagen from the solution, centrifugation is used at 20,000 g for 1 h. A refrigerated
centrifuge is used in order to maintain the required temperature. To further purify the
collagen sample, it can then be redissolved in 0.5 M acetic acid and then separated
by repeated dialysis. The collagen extract now needs to be dried for more effective
storage and packaging. For this mild drying method is required as excessive heat
will destroy the triple helix structure and the resulting extract will be hydrolyzed
collagen. Freeze drying can be employed here. It is commonly used in such cases
where a delicate sample needs to be dried. A temperature between 4 and 8 °C is
maintained throughout the pretreatment and extraction to prevent degradation of the
collagen.
The enzyme extraction can be carried out using the residue from the acid extraction
(Mahboob 2015). The residue contains the collagen proteins which do not dissolve
in acid but can be dissolved in pepsin. This method allows obtaining two types of
collagen from the same source thus making optimal use of the fish waste. The pH is
adjusted to that suitable for the enzyme by soaking in 0.5 M acetic acid in a solid to
liquid ratio of 1:15. The pepsin is then added and continuously stirred for a duration
of 48 h. A low temperature is also maintained at 4 °C. At the end of the extraction,
the solid residue is then separated from the liquid which contains the collagen. The
collagen is precipitated using sodium chloride, and the precipitated collagen is then
separated from the salt solution and further purification by repeated dialysis then
follows.
The type of extraction process has some effect on the collagen yield and properties.
For example, acid extraction from scales of goatfish gave collagen yield of 0.46%
while pepsin extraction gave a yield of 1.20% (dry weight basis). The acid-soluble
collagen had slightly higher glass transition temperature of 41.58 °C while that of
pepsin soluble collagen was 41.01 °C (Matmaroh et al. 2011). Sodium hydroxide
extraction results in mostly denatured collagen or hydrolyzed collagen which is a
mixture of polypeptides of different chain lengths. The extraction process therefore
12 Collagen
has been washed off and the system is neutral. The type of water used depends on the
level of purity required. Clean tap water could be used, or distilled water or ultrapure
water.
For the acid-based extraction, the fish waste was treated with acid in order to
dissolve out the acid-soluble collagen from the rest of the tissue. The first step was to
remove the fat using 10% butyl alcohol at a 1:10 ratio of solid to alcohol for a duration
of 8 h. The fats are alcohol soluble so it will be removed from the fish waste. The fish
waste is then washed with cold water to remove any residue of alcohol and fat from the
surface. The next stage is then to extract the collagen in acetic acid at a concentration
of 0.5 M and 1:15 solid to liquid ratio. The extraction is allowed a duration of 24 h. The
extraction time can be varied for different samples since the composition of collagen
and other components varies for different types of fish. Defatting prior to extraction
prevents the fat from being released alongside the collagen during the extraction. The
fat will dissolve in alcohol but collagen will not, however the fat could be released
into the acetic acid solution hence the order is also important. The collagen dissolved
in acetic acid is then precipitated out in a 0.05 M tris (hydroxymethyl) aminomethane
containing sodium chloride at a concentration of 2.6 M. To separate the precipitated
collagen from the solution, centrifugation is used at 20,000 g for 1 h. A refrigerated
centrifuge is used in order to maintain the required temperature. To further purify the
collagen sample, it can then be redissolved in 0.5 M acetic acid and then separated
by repeated dialysis. The collagen extract now needs to be dried for more effective
storage and packaging. For this mild drying method is required as excessive heat
will destroy the triple helix structure and the resulting extract will be hydrolyzed
collagen. Freeze drying can be employed here. It is commonly used in such cases
where a delicate sample needs to be dried. A temperature between 4 and 8 °C is
maintained throughout the pretreatment and extraction to prevent degradation of the
collagen.
The enzyme extraction can be carried out using the residue from the acid extraction
(Mahboob 2015). The residue contains the collagen proteins which do not dissolve
in acid but can be dissolved in pepsin. This method allows obtaining two types of
collagen from the same source thus making optimal use of the fish waste. The pH is
adjusted to that suitable for the enzyme by soaking in 0.5 M acetic acid in a solid to
liquid ratio of 1:15. The pepsin is then added and continuously stirred for a duration
of 48 h. A low temperature is also maintained at 4 °C. At the end of the extraction,
the solid residue is then separated from the liquid which contains the collagen. The
collagen is precipitated using sodium chloride, and the precipitated collagen is then
separated from the salt solution and further purification by repeated dialysis then
follows.
The type of extraction process has some effect on the collagen yield and properties.
For example, acid extraction from scales of goatfish gave collagen yield of 0.46%
while pepsin extraction gave a yield of 1.20% (dry weight basis). The acid-soluble
collagen had slightly higher glass transition temperature of 41.58 °C while that of
pepsin soluble collagen was 41.01 °C (Matmaroh et al. 2011). Sodium hydroxide
extraction results in mostly denatured collagen or hydrolyzed collagen which is a
mixture of polypeptides of different chain lengths. The extraction process therefore