🍎 Hydroxyproline (collagen) in meat — hydrolysis in sulfuric acid, oxidation with chloramine T, photometry at 558 nm
In short
About 4 g of the sample is hydrolysed for 16 h at 105 °C in sulfuric acid 3 mol/l; after filtration and dilution the hydroxyproline is oxidised with chloramine T, coupled with p-dimethylaminobenzaldehyde at 60 °C and the absorbance of the red dye is measured at 558 nm against a calibration curve. The test is performed according to PN-ISO 3496:2000; Scope: Meat and products with a hydroxyproline content < 0.5 % (m/m). The procedure comprises 10 steps (about 17 h 10 min in total); it is used for: Determination of the content of collagen (connective tissue) in meat and meat products, including poultry ones, Assessment of the quality of the meat raw material and of the products (the ratio of collagen to protein), Studies of the tenderness and the solubility of intramuscular collagen (total collagen = hydroxyproline × 7.25).
At a glance
- Standard: PN-ISO 3496:2000
- Category: Food chemistry
- Procedure steps: 10
- Total time of stages: 17 h 10 min
- Scope: Meat and products with a hydroxyproline content < 0.5 % (m/m)
- Hydrolysis: 30 ± 1 ml of H2SO4 3 mol/l, 16 h at 105 °C
- Colour reaction: Chloramine T 20 ± 1 min at room temperature; the colour reagent 20 min at 60 °C ± 0.5 °C
Overview
The standard specifies a method of determining the content of hydroxyproline in all kinds of meat and meat products, including poultry, with a hydroxyproline content below 0.5 % (m/m). Hydroxyproline is an amino acid characteristic of collagen, and for that reason its content serves to calculate the content of connective tissue: in studies of beef muscles the hydroxyproline determined is converted into total collagen with the factor 7.25. The description is based on the preview of ISO 3496:1994, which PN-ISO 3496:2000 implements.
The method consists of the hydrolysis of the test portion in sulfuric acid at 105 °C, the filtration and dilution of the hydrolysate, the oxidation of the hydroxyproline with chloramine T and the formation of a red compound with p-dimethylaminobenzaldehyde, whose absorbance is measured at 558 nm. The concentration is read off from a calibration curve prepared for every series of analyses from four standard solutions of hydroxyproline (0.5; 1; 1.5 and 2 µg/ml).
The content of collagen matters for the tenderness of meat and the quality of products: in beef muscles the content of total collagen ranged from 0.4 % (longissimus dorsi) to 1.3 % (infraspinatus), and hydroxyproline figures in the list of determinations of the QAFP branch booklet for canned meat.
Method principle
About 4 g of the sample (weighed to the nearest 0.001 g) is hydrolysed with 30 ± 1 ml of sulfuric acid 3 mol/l in a drying oven at 105 °C for 16 h (conveniently overnight). The hot hydrolysate is filtered into a 250 ml volumetric flask, washed three times with 10 ml of hot acid and made up with water. A portion V of the hydrolysate is diluted to 250 ml so that the concentration of hydroxyproline falls within the range 0.5–2 µg/ml (usually V = 5–25 ml depending on the amount of connective tissue). To 4.00 ml of the diluted solution 2.00 ml of the chloramine T reagent is added (20 min at room temperature), then 2.00 ml of the colour reagent with p-dimethylaminobenzaldehyde, it is heated for exactly 20 min in a bath at 60 °C, cooled and after 30 min the absorbance is measured at 558 ± 2 nm in a 10 mm cell against water. The content of hydroxyproline: wh = 6.25·c/(m·V) [% by mass], where c is the concentration from the calibration curve (µg/ml), m the mass of the test portion (g), V the volume of the portion of the hydrolysate (ml). The result is given to the nearest 0.01 %.
Applications
- Determination of the content of collagen (connective tissue) in meat and meat products, including poultry ones
- Assessment of the quality of the meat raw material and of the products (the ratio of collagen to protein)
- Studies of the tenderness and the solubility of intramuscular collagen (total collagen = hydroxyproline × 7.25)
- Control of canned meat — the determination is in the list of procedures of the QAFP system
Key parameters
| Parameter | Value |
|---|---|
| Scope | Meat and products with a hydroxyproline content < 0.5 % (m/m) |
| Hydrolysis | 30 ± 1 ml of H2SO4 3 mol/l, 16 h at 105 °C |
| Colour reaction | Chloramine T 20 ± 1 min at room temperature; the colour reagent 20 min at 60 °C ± 0.5 °C |
| Measurement | 558 ± 2 nm, glass cell 10 mm, against water; the blank should not exceed an absorbance of 0.040 |
| Calibration curve | Four solutions of 0.5; 1; 1.5 and 2 µg/ml of hydroxyproline, a new one for every series |
| Precision | r = 0.0131 + 0.0322·wh; R = 0.0195 + 0.0529·wh (wh — the mean, % by mass) |
Standard
- Standard number
- PN-ISO 3496:2000
- Title (PL)
- Mięso i przetwory mięsne — Oznaczanie zawartości hydroksyproliny
- Title (EN)
- Meat and meat products — Determination of hydroxyproline content
Step-by-step procedure
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Preparation of the sample
Cut raw meat cold into cubes of about 8 mm side, close it hermetically or pack it under vacuum and heat it so as to maintain at least 70 °C for at least 30 min (this softens the connective tissue); cool it and homogenise it in a mincer. Homogenise cooked products directly. Analyse within 24 h, taking care that the fat and the liquid are evenly distributed.
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Test portion
Weigh out about 4 g of the sample to the nearest 0.001 g into a hydrolysis flask; make sure that the material does not stick to the walls.
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Hydrolysis
Add 30 ± 1 ml of sulfuric acid 3 mol/l, cover with a watch glass and put it into a drying oven at 105 °C for 16 h (conveniently overnight).
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Filtration and dilution of the hydrolysate
Filter the hot hydrolysate into a 250 ml volumetric flask; wash the flask and the filter paper three times with portions of 10 ml of hot sulfuric acid 3 mol/l, add the washings, make up with water to the mark and mix.
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Working dilution
With a pipette transfer into a 250 ml flask a volume V of the hydrolysate chosen so that after the dilution the concentration of hydroxyproline is 0.5–2 µg/ml (most often V = 5–25 ml, depending on the amount of connective tissue); make up with water.
-
Oxidation with chloramine T
Transfer 4.00 ml of the diluted solution into a test tube, add 2.00 ml of the chloramine T reagent, mix and leave for 20 ± 1 min at room temperature.
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Colour reaction
Add 2.00 ml of the colour reagent, mix thoroughly, close the test tube with aluminium or plastic foil and put it immediately into a water bath at 60 °C for exactly 20 min.
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Cooling and measurement
Cool the test tube under running water for at least 3 min, leave it for 30 min at room temperature and measure the absorbance at 558 ± 2 nm in a 10 mm glass cell against water. Subtract the absorbance of the blank (a double one, with water instead of the hydrolysate).
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Calibration curve
Carry out operations 6–8 with 4.00 ml of each of the four standard solutions; plot the absorbance (after the subtraction of the blank) against the concentration and draw the best-fitting straight line through the points and the origin. Prepare a new curve for every series of analyses.
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Calculation
wh = 6.25·c/(m·V), where c is the concentration of hydroxyproline in the diluted hydrolysate (µg/ml), m the mass of the test portion (g), V the volume of the portion of the hydrolysate (ml). Give the result to 0.01 %; discard both results and repeat the determination where the difference exceeds r = 0.0131 + 0.0322·wh.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Electric mincer | With horizontal knives, high-speed | — |
| Hydrolysis flasks | Round- or flat-bottomed, wide-necked, about 200 ml, covered with watch glasses of 5–6 cm | — |
| Drying oven | Controlled at 105 ± 1 °C | — |
| Water bath | Maintaining 60 ± 0.5 °C | — |
| Spectrometer or photoelectric colorimeter | For the measurement at 558 ± 2 nm; glass cells with an optical path of 10 mm | — |
| Analytical balance, pH meter, 250 ml volumetric flasks, filter papers | An accuracy of weighing of 0.001 g; filter papers of 12.5 cm diameter | — |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Sulfuric acid, solution 3 mol/l | 7664-93-9 | Add 320 ml of the concentrated acid (density 1.84 g/ml) to 750 ml of water in a 2 l flask, cool it and make up to the mark |
| Buffer pH 6.8 | — | Dissolve 26.0 g of citric acid monohydrate, 14.0 g of sodium hydroxide and 78.0 g of anhydrous sodium acetate in 500 ml of water, add 250 ml of propan-1-ol and make up to 1 l; stable for several weeks at 4 °C in the dark |
| Chloramine T (the sodium salt of N-chloro-p-toluenesulfonamide, trihydrate) | 127-65-1 | Dissolve 1.41 g in 100 ml of the buffer; prepare immediately before use |
| p-Dimethylaminobenzaldehyde | 100-10-7 | The colour reagent: dissolve 10.0 g in 35 ml of perchloric acid 60 % (m/m) and slowly add 65 ml of propan-2-ol; prepare on the day of use; where necessary purify it by crystallisation from 70 % ethanol |
| Perchloric acid 60 % (m/m) | 7601-90-3 | A component of the colour reagent |
| Hydroxyproline (trans-4-hydroxy-L-pyrrolidine-2-carboxylic acid) | 51-35-4 | Stock solution: 50 mg in 100 ml of water with a drop of sulfuric acid (stable for at least 1 month at 4 °C); on the day of use dilute 5 ml to 500 ml, and from that 10, 20, 30 and 40 ml to 100 ml — the standards of 0.5; 1; 1.5 and 2 µg/ml |
Health and safety (OHS)
- Sulfuric acid 3 mol/l and the concentrated acid (the preparation of the solution) — goggles, gloves, a laboratory coat; add the acid to the water, filter the hot hydrolysate carefully
- The perchloric acid 60 % in the colour reagent is a strong oxidising agent — do not allow contact with organic materials outside the procedure, work under a fume hood
- p-Dimethylaminobenzaldehyde and chloramine T — avoid inhaling the dust and contact with the skin
- A drying oven at 105 °C for 16 h — the flasks covered with watch glasses, take them out with thermal gloves
Frequently asked questions
Which standard describes this test?
The test is performed according to PN-ISO 3496:2000 — “Meat and meat products — Determination of hydroxyproline content”.
How does this method work?
About 4 g of the sample (weighed to the nearest 0.001 g) is hydrolysed with 30 ± 1 ml of sulfuric acid 3 mol/l in a drying oven at 105 °C for 16 h (conveniently overnight).
What is the measuring range and accuracy?
Scope: Meat and products with a hydroxyproline content < 0.5 % (m/m); Hydrolysis: 30 ± 1 ml of H2SO4 3 mol/l, 16 h at 105 °C; Colour reaction: Chloramine T 20 ± 1 min at room temperature; the colour reagent 20 min at 60 °C ± 0.5 °C; Measurement: 558 ± 2 nm, glass cell 10 mm, against water; the blank should not exceed an absorbance of 0.040.
How long does the test take?
The times given for the individual stages add up to approximately 17 h 10 min. The procedure comprises 10 steps.
What equipment is required?
Electric mincer, Hydrolysis flasks, Drying oven, Water bath, Spectrometer or photoelectric colorimeter, Analytical balance, pH meter, 250 ml volumetric flasks, filter papers.
Where is this test used?
Determination of the content of collagen (connective tissue) in meat and meat products, including poultry ones; Assessment of the quality of the meat raw material and of the products (the ratio of collagen to protein); Studies of the tenderness and the solubility of intramuscular collagen (total collagen = hydroxyproline × 7.25); Control of canned meat — the determination is in the list of procedures of the QAFP system.
What safety precautions apply?
Sulfuric acid 3 mol/l and the concentrated acid (the preparation of the solution) — goggles, gloves, a laboratory coat; add the acid to the water, filter the hot hydrolysate carefully; The perchloric acid 60 % in the colour reagent is a strong oxidising agent — do not allow contact with organic materials outside the procedure, work under a fume hood; p-Dimethylaminobenzaldehyde and chloramine T — avoid inhaling the dust and contact with the skin; A drying oven at 105 °C for 16 h — the flasks covered with watch glasses, take them out with thermal gloves.
Which laboratory can perform this test?
The test is performed by laboratories accredited to ISO/IEC 17025. On LabCoda you can find them by the standard number PN-ISO 3496.