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Determination of Soluble and Total Collagen
Transcript of Determination of Soluble and Total Collagen
Determination of the Collagen
Main structural protein in the
Mechanical resistance to stretching.
Substance that holds the whole
Several type of collagen.
Pulverized meat samples (4 g).
Suspend with 12 ml of strength Ringer’s solution.
Heat in the water bath
Every 10 min
Let for 30 min at room temperature
6000 X G
Remove supernatant into 50-ml screw top test tubes with soluble collagen
Suspend with strength Ringer’s solution
Remake the centrifugation and the supernatant removal
Add dd H2O into test tube
Pour into 50-ml screw top test tubes
Add concentrated hydrochloric acid to centrifuge tubes
Pour into test tubes
Add concentrated HCl to supernatant tube
Separation of soluble and Insoluble Collagen to obtain hydroxyproline
depends on many factors
- rearing conditions
Add aliquots of sample into screw top tubes
Add isopropanol to tube and vortex
Add oxidant solution in the tube
Let at room temperature
for 4 min
Add Erlich’s reagent to the tube and close this one.
Heat tubes in the water bath
Cool tubes in running tap water
Determination of hydroxyproline
Collagen Analysis Results
Exemple of regression
Determination of hydroxyproline content
Convert hydroxyproline to collagen (μg collagen/g of sample)
multiply the supernatants by 7.52
multiply the residuals by 7.25
masse of hydroxyproline x dilution factor
masse of sample
Add methyl red indicator
neutralize the solution
5.0 N NAOH to a yellow endpoint.
filter through Whatman N°1
supernatants to 100 ml
residuals to 500 ml
With dd H2O
12 h ,120-127°,20 + 2 lbs
Add carbon decolorizing agent
200 mg to the supernatant
900 mg to the residuals
mix and filter through Whatman N°1