6.5 Extraction of Carrageenan
131
6.5.2 Enzyme Extraction
Carrageenan can also be isolated from red algae using enzymatic digestion. This
involves breaking down the proteins within the structure to release the polysaccharides. The sulfated polysaccharide, which in red algae is carrageenan, is then purified
through precipitation. This method has been used by Souza et al. (2018) to extract
carrageenan from the H. musciformis red algae. In the said study, the harvested
biomass was cleaned and washed to remove epiphytes and other unwanted matter.
The process of extraction then begins with proteolytic digestion using the protease
enzyme, papain from the papaya fruit (Carica papaya). This breaks down the protein into smaller units of peptides and amino acids. The digestion was carried out
at 60 °C in a sodium acetate buffer at pH 5 containing 5 mM of EDTA and cysteine. The digestion was allowed a period of 6 h. The next stage is then to isolate
the freed sulfated polysaccharide from the solution. This was done by precipitation
with cetylpyridinium chloride. This method resulted in a carrageenan yield of 28%
carrageenan per gram of dry red algae biomass. The carrageenan extracted showed
17.3% free sulfate content and a very polydisperse extract with peak molar mass of
519.1 kDa measured using gel permeation chromatography.
6.5.3 Semi-refined Carrageenan
The semi-refined form of carrageenan also finds some use. This form of carrageenan
is one, whereby other water-soluble components of the carrageenan are removed by
dissolving them of at lower water temperature such that the carrageenan alongside
the salt still remains behind in the seaweed. This process requires much lower cost
of processing and is used where the presence of cellulose alongside the carrageenan
does not affect the application. The seaweed is washed to get rid of debris. It is
then treated in a solution of potassium hydroxide at a moderately high temperature.
This allows the reduction in degree of sulfation as well as the hardening of the gel
structure, while the carrageenan is still within the seaweed biomass. The heat allows
the aqueous solution of alkali to penetrate the cell walls allowing interaction between
the OH- ions and the sulfate groups and the K+ ions and the gel networks resulting in
a stronger gel which does not dissolve in the solution. The other components of the
seaweed such as proteins, water-soluble carbohydrates and other smaller molecules,
however, are removed in this process. This leaves behind a solid mass which is
mostly carrageenan and cellulose. This can be heat sterilized and used as low-grade
gelling agent in canned pet food where a cheaper alternative to refined carrageenan is
acceptable. Alternatively, it can be sold to carrageen manufacturers as feedstock for
producing refined carrageenan. The latter option enables value addition to seaweed
from place of origin and reduces the cost of transportation and waste treatment for
the manufacturer. When semi-refined carrageenan is produced using more stringent
131
6.5.2 Enzyme Extraction
Carrageenan can also be isolated from red algae using enzymatic digestion. This
involves breaking down the proteins within the structure to release the polysaccharides. The sulfated polysaccharide, which in red algae is carrageenan, is then purified
through precipitation. This method has been used by Souza et al. (2018) to extract
carrageenan from the H. musciformis red algae. In the said study, the harvested
biomass was cleaned and washed to remove epiphytes and other unwanted matter.
The process of extraction then begins with proteolytic digestion using the protease
enzyme, papain from the papaya fruit (Carica papaya). This breaks down the protein into smaller units of peptides and amino acids. The digestion was carried out
at 60 °C in a sodium acetate buffer at pH 5 containing 5 mM of EDTA and cysteine. The digestion was allowed a period of 6 h. The next stage is then to isolate
the freed sulfated polysaccharide from the solution. This was done by precipitation
with cetylpyridinium chloride. This method resulted in a carrageenan yield of 28%
carrageenan per gram of dry red algae biomass. The carrageenan extracted showed
17.3% free sulfate content and a very polydisperse extract with peak molar mass of
519.1 kDa measured using gel permeation chromatography.
6.5.3 Semi-refined Carrageenan
The semi-refined form of carrageenan also finds some use. This form of carrageenan
is one, whereby other water-soluble components of the carrageenan are removed by
dissolving them of at lower water temperature such that the carrageenan alongside
the salt still remains behind in the seaweed. This process requires much lower cost
of processing and is used where the presence of cellulose alongside the carrageenan
does not affect the application. The seaweed is washed to get rid of debris. It is
then treated in a solution of potassium hydroxide at a moderately high temperature.
This allows the reduction in degree of sulfation as well as the hardening of the gel
structure, while the carrageenan is still within the seaweed biomass. The heat allows
the aqueous solution of alkali to penetrate the cell walls allowing interaction between
the OH- ions and the sulfate groups and the K+ ions and the gel networks resulting in
a stronger gel which does not dissolve in the solution. The other components of the
seaweed such as proteins, water-soluble carbohydrates and other smaller molecules,
however, are removed in this process. This leaves behind a solid mass which is
mostly carrageenan and cellulose. This can be heat sterilized and used as low-grade
gelling agent in canned pet food where a cheaper alternative to refined carrageenan is
acceptable. Alternatively, it can be sold to carrageen manufacturers as feedstock for
producing refined carrageenan. The latter option enables value addition to seaweed
from place of origin and reduces the cost of transportation and waste treatment for
the manufacturer. When semi-refined carrageenan is produced using more stringent