Comparative Biochemical
properties of collagen from
skin and bones of black
drum (Pogonia cromis)
Jack N. Losso
Department of Food Science
Louisiana State University Agricultural Center
Baton Rouge, LA 70803
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Collaborators
• Masahiro Ogawa
• Jon Bell
• Mark Schexnayder
• Mike Moody
• Ralph Portier
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Presentation Overview
• Introduction
• Marine Collagen and Rationale for Research
• Methodologies
• Results
• Future Prospects
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Introduction
• By 2025 more food and products for human
health will be originating from the sea (USDA
estimates)1
• Need for efficient and full utilization of harvested
aquatic-based food resources
• In 2000, domestic landings of fish and shellfish
in the Gulf States and adjacent waters were 0.8
million metric tons, and accounted for about
20% of the total landings in the U.S.
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Introduction
• Abundant coastal and marine resources in Louisiana
• Louisiana ranks second to Alaska in total landings
• Louisiana seafood processors generate thousands of
tons of aquatic-based food waste annually
• Compliance with environmental laws
• Potentials of and interest in obtaining health enhancing
products from aquatic sources
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Value-added from marine
by-products…
• Functional foods (Calcium, oil,
antioxidants, color, caviar)
• Fine biochemicals (enzymes, proteins,
nucleic acids)
• Pharmaceuticals (antimicrobials, antiinflammatory,
enzyme
inhibitors)
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Marine Collagen…
• Most abundant protein in vertebrates (~25% of total)
• Major structural element of skin, bones, tendons,
cartilage, blood vessels, and teeth
• Structure distinguished by the formation of a right-
handed triple superhelical rod consisting of three almost
identical polypeptide chains consisting of repeating
triplets, (Gly-X-Y)n, where X and Y are often proline
(Pro) or hydroxyproline (Hyp)
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Marine Collagen…
• Collagen from land-based animals (bovine and
porcine) used in functional food, cosmetics and
pharmaceuticals
• Emergence of bovine spongiform
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encephalopathy (BSE), foot-and-mouth disease
(FMD) crisis calls into question the use of
collagen and collagen-derived products from
land-based animal skin
Marine Collagen…
• Fish skin: high source of collagen
• However, fish species with reported
collagen denaturation temperatures above
30 ºC are very limited (e.g., skipjack and
carp)
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Marine Collagen…
• Collagen molecules in solution denature close to
the upper limit of physiological temperature or
the maximum body temperature of the animal
species from which the collagen is extracted
• Tropical and subtropical fish in the Gulf States
may contain collagen with denaturation
temperatures above 30 ºC
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Research Objective…
• The objective of this research was to
compare the biochemical properties
and thermal stability of collagen from
black drum skin and bones as an
alternate of land-based animal collagen
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Fish Skin
Collagen
Preparation
Fish Skin Fish Skin
Fish Skin
Supernatant
Supernatant
Collagen 1
Collagen 1
Alkaline treatment
Alkaline treatment
Extraction with 0.05MCH3COOH
Extraction with 0.05MCH3COOH
Residue
Residue
Acid/Pepsin Extract
Acid/Pepsin Extract
Collagen 2
Collagen 3
Collagen 2
Collagen 3 Supernatant
Supernatant
Residue
Residue
Salt precipitation
Salt precipitation
Collagen 1
2
Collagen 1 Collagen
Collagen 2
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Collagen 3
Collagen 3
Results…
γ
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β
200 kDa→
α1
α2
116 kDa→
Bone
Skin
SDS-PAGE of black drum collagen
Amino Acid Composition of Black
drum Collagen
Skin
ASC
Hyp 80.2 7.0
Pro 119.6 2.7
Gly 319.8
Ala 120.7
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Bones
PSC
89.7 6.2
107.4
ASC
80.2 2.2
PSC
84.6 1.0
21.3 101.1
1.3
98.8 314.6
7.4
12.2 338.0
10.5
2.4
18.8 126.4
1.2
1.6
130.8
342.0
106.6
129.8
2.3
Circular dichroism…
• CD was measured with
3 mg/ml collagen in 0.1
M acetic acid.
• Scans (5) obtained
between 250-195 nm
using mean amino acid
residue = 91 g/mol
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CD spectra of (a) black drum bone and scale collagen
The spectra were
taken at 15 °C. Filled
diamond, black drum
ASC; unfilled
diamond, black drum
PSC; filled triangle,
sheepshead ASC;
unfilled triangle, PSC,
sheepshead PSC.
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Wavelength (nm)
20000
10000
205
-10000
225
-30000
-40000
-50000
-60000
-70000
235
245
Denatured collagen
(45 ºC, 30min)
0
Native collagen (15 ºC)
CD spectra of black drum skin collagen
10000
8000
6000
4000
2000
-2000
-4000
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215.
- 20000
[θ]220 (deg cm2 dmol-1)
[θ] (deg cm2 dmol-1)
CD and melting characteristics of black drum collagen
10
20
30
40
50
Temperature (ºC)
Melting curve of black drum skin collagen
Thermal denaturation
of skin collagen
ASC
PSC
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Thermal denaturation curves of bone
collagen measured by viscosity
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Unfilled diamond w/ broken line = black drum; unfilled
triangle = sheepshead
Metling curve determined
by measuring molar
ellipticity at λ of positive
extreme (220 nm) while
heating from 15 to 45 ºC.
Transition temperature Tm
was determined.
Melting curves of
collagens from (a) bones
and (b) scales.
The fraction change of
[θ]220 was plotted against
temperature.
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Biochemical properties of black drum collagen
• Secondary structure
• Denaturation temperature
• CD spectrum of the
• Transition temperature Tm for
native ASC sample
showed that the ASC
formed collagen triple
helices
• The structure was lost at
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45 ºC
black drum ASC was 34.8 ºC,
which is similar to that of calf
hide ASC (36.3 ºC)
Imino Acids Content of Black drum
Collagen
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Source of
collagen
Imino
acids
Tm-CD ºC Td-V ºC
Skin ASC
199.8
34.8
34.2
Skin PSC
197.1
35.1
35.8
Bone ASC
193.4
35.1
35.3
Bone PSC
191.2
34.9
35.7
Conclusion…
• Black drum… an excellent source of
collagen
• Isolated collagen has properties similar to
land-based animal collagen
• Biomedical applications are awaiting
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Future Prospects
• Biomedical applications in
areas such as
- Anti-inflammatory
- Inhibition of angiogenesis
-Tissue Engineering
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THANK YOU
QUESTIONS???
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