A standardised static<i>in vitro</i>digestion method suitable for food – an international consensus

<p>The paper presents an international consensus for a standardised static<italic>in vitro</italic>digestion method for food.</p>

section_content: Prepare 100 mL of a 20 mM sodium phosphate buffer containing 6. 7 mM NaCl. Adjust it to pH 6. 9 at 20°C with 1 M NaOH. Prepare a 1. 0% w/v soluble starch solution in the sodium phosphate buffer pH 6. 9 by dissolving 0. 25 g soluble potato starch (for instance ref S2630 Sigma-Aldrich) in an initial volume of 20 mL of buffer. Cover the beaker to avoid water evaporation and bring to boil while stirring and maintain just below boiling temperature for 15 minutes. After cooling to room temperature, complete the starch solution to the appropriate volume (25 mL) by adding 5 mL of purified water. 

Preparation of colour reagent solution: Prepare a 5. 3 M sodium potassium tartrate solution in 2 M NaOH. Dissolve 0. 8 g NaOH in 10 mL purified water. Heat the solution to reach a temperature ranging between 50 to 70°C. Dissolve 12. 0 g of sodium potassium tartrate tetrahydrate (MW: 282. 22 g/mol), in 8. 0 mL of warm 2 M NaOH solution. Maintain at this temperature while constant stirring to dissolve but do not boil. 

Prepare a 96 mM 3,5-dinitrosalicylic acid (MW: 228. 12 g/mol) solution by dissolving 438 mg of acid in 20 mL of purified water. Heat the solution to reach a temperature ranging between 50 to 70°C. Maintain at this temperature while constant stirring to dissolve but do not boil. Heat 12 mL of purified water to 60°C, add slowly 8 mL of the 5. 3 M Sodium Potassium Tartrate Solution. Then add 20 mL of the 96 mM 3,5-dinitrosalicylic acid solution. Stir until complete dissolution. The solution can be stored up to 6 months in amber flask at room temperature. Prepare 10 mL of 0. 2 % w/v maltose standard (for instance ref M5885 Sigma-Aldrich). 

Enzyme: Just before use, prepare a solution containing approximately 1 unit/mL of α-amylase in purified water.

section_content: Set the spectrophotometer at 540 nm and 20°C. Set a bench top shaking incubator fitted with sample holder at 20°C. Heat a water bath at 100°C, fit it with a sample holder to stop the reaction. Prepare a container filled with ice to cool the samples. 

Pipette into 15 mL cap covered containers 1 mL of substrate (solution of potato starch), mix by swirling and incubate at 20°C for 3-4 minutes to achieve temperature equilibration. 

Test: Then add 500 µl to 1 mL of enzyme solution, mix by swirling and incubate at 20°C for exactly 3 minutes. After 3 minutes, stop the solution by adding 1 mL of colour reagent solution, cap the reaction vessel, place in the boiling water bath, complete the volume of enzyme added to 1 mL (for instance if 700 µL has been added, add 300 µL of enzyme solution). 

Boil for exactly 15 minutes and cool on ice for a few minutes. Add 9 mL of purified water. Mix by inversion. Pipette 3 mL of the reaction mixture in a cuvette of spectrophotometer and record the absorbance at 540 nm, noted A 540 Test. 

Blank: For blank assays, the protocol is similar but no enzyme is added before the 3 minute incubation. It is only after the addition of the colour reagent and after putting the reaction vessel in the boiling bath, that 1 mL of enzyme solution is added. 

After blank procedure, the absorbance, A 540 Blank is similarly recorded. 

To test 3 concentrations of enzymes, the following tests can be run successively: Method: Spectrophotometric Stop Reaction
section_name: Assay:

section_content: Haemoglobin + H 2 O pepsin > TCA soluble tyrosine containing peptides Unit definition: One unit will produce a A 280 of 0. 001 per minute at pH 2. 0 and 37°C, measured as TCA-soluble products. 

These units are often referred to "Sigma" or "Anson" pepsin units. 

Conditions: T = 37°C, pH = 2. 0, A 280nm, light path = 1 cm
section_name: Principle:

section_name: Protocol

section_content: Substrate: a 2 % w/v haemoglobin (bovine blood haemoglobin, ref H2500 Sigma-Aldrich) solution is prepared by dispersing 0. 5 g haemoglobin in 20 mL purified water and then diluting this solution in 5 mL of 300 mM HCl in order to get a solution at 2 % w/v haemoglobin at pH 2. 

Enzyme: A stock solution of Pepsin (porcine pepsin, ref P6887 Sigma-Aldrich) is dissolved in 150 mM NaCl adjusted to pH 6. 5 using 100 mM NaOH. The stock solution has to be stored on ice or refrigerated at 4°C. Just before the assay, a range of 5 to 10 concentrations of pepsin in 10 mM HCl has to be prepared, we suggest for instance to dilute the pepsin at: 5, 10, 15, 20, 25, 30 µg/mL.
section_name: Preparation of reagents

section_content: Set the spectrophotometer at 280 nm and 20°C. Set a bench top shaking incubator fitted with a sample holder at 37°C. 

Test: Pipette 500 µl of haemoglobin solution into 2 mL eppendorf and incubate in shaking incubator at 37°C for 3-4 minutes to achieve temperature equilibration. 

Add 100 µl of pepsin solutions for each concentration which is tested and incubate for 10 minutes exactly. To stop the reaction, 1 mL of TCA (5% w/v trichloroacetic acid) is added in each tube. In order to get a clear soluble phase available for absorbance measurement, a centrifugation step is useful. Place eppendorf in a centrifuge at 6,000 g during 30 minutes to precipitate haemoglobin. 

Pipette into quartz cuvettes a mix of 2. 6 mL of 46 mM Tris/HCl buffer (pH 8. 1) and 0. 3 mL of the substrate (10 mM TAME), mix by inversion and incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibration. 

Test: Then add 100 µl of trypsin solutions for each concentration which is tested and record the absorbance increase at 247 nm, noted ΔA 247, during 10 min in continuum until levelling off. Determine the slope ΔA 247 Test from the initial linear portion of the curve. If no linear part is found repeat the test using less enzyme. 

Blank: For blank assays, the protocol is similar but no enzyme is added and the absorbance is similarly recorded, however it stabilises quicker (within 5 min). ΔA 256 Blank is obtained, it should be close to zero.
section_name: Assay:

section_content: Obtain the slopes ΔA 247 [unit absorbance/minute] for both the blank and test reactions using the maximum linear rate and over at least 5 minutes:

(540 * ) Alternatively, precisely diluted commercial NaOH stock solutions can be used. 

Enzyme: Dissolve 5 mg of enzyme powder in 5 mL of purified water. Store on ice. At least 2 amounts of enzyme solution

(1 mg/mL) have to be tested.
section_name: Calculations:

section_content: Switch on a thermo-regulated pHstat device, such as Tritino or Dosino from Metrohm, fitted with a jacketed and capped reaction vessel and mechanical stirrer. 

Blank: Pour into the titration vessel 15 mL of the aqueous phase and add 0,5 mL of tributyrin. Make sure the volume of the assay is high enough to ensure adequate pH-measurement (microelectrode should be correctly immersed). Switch on magnetic stirring (tributyrin will get dispersed) and let equilibrate at 37°C. 

Monitor the rate of titrant solution (NaOH) delivery which is required to maintain a pH of 8 at 37°C. 

Test: Pour into the titration vessel 14. 5 mL of the assay solution and add 0. 5 mL of tributyrin. Ensure to use the correct flask to ensure adequate pH-measurement (microelectrode should be correctly immersed). Switch on mechanical stirrer (tributyrin will be dispersed) and let equilibrate at 37°C. 

Add 10 to 100 µL of a diluted solution of pancreatic lipase containing colipase. If the lipase powder does not contain colipase make sure colipase is added in functional excess (colipase/lipase molar ratio 2:1) before adding the enzyme to allow measuring maximum activity. Monitor the rate of titrant solution (NaOH) delivery, which is required to compensate for the liberation of free fatty acids and maintain a pH of 8 at 37°C. These conditions allow measuring linear kinetics of free fatty release for at least 5 minutes. Check that the same activity (U/mg) is obtained for the both tested enzyme amounts.
section_name: Assay:

section_name: Calculations

section_content: Remove the soluble phase and place it in quartz cuvettes and read the absorbance at 280 nm, A 280 Test. 

Blank: For blank tests, the same procedure is followed but the pepsin is added after the addition of TCA, which stops the reaction. The blank absorbance is noted A 280 Blank. 

During the assay, a linear curve has to be obtained. The absorbance is a function of the pepsin concentration. If no linear part is found, it can be due to a large amount of enzyme, therefore it is necessary to use a more dilute enzyme solution. Prepare a 46 mM TRIS/HCl buffer, containing 11. 5 mM CaCl 2 adjust its pH at 8. 1 at 25°C.

section_content: Set the spectrophotometer at 247 nm and 25°C.
section_name: Assay:

section_content: Set the spectrophotometer at 256 nm and 25°C. 

Pipette into quartz cuvettes a mix of 1. 5 mL of Tris/HCl buffer and 1. 4 mL of substrate (BTEE), mix by inversion and incubate in spectrophotometer at 25°C for 3-4 minutes to achieve temperature equilibration. 

Test: Then add 100 µl of chymotrypsin and record the absorbance increase at 256 nm during 10 min in continuum until levelling off. Determine the slope ΔA 256 Test from the initial linear portion of the curve. If no linear part is found repeat the test using less enzyme. 

Blank: For blank assays, the protocol is similar but no enzyme is added and the absorbance is similarly recorded but get stable very quickly (within 5 min). ΔA 256 Blank is obtained, it should be close to zero.
section_name: Assay:

section_content: Obtain the slopes ΔA 256 [unit absorbance/minute] for both the blank and test reactions using the maximum linear rate and over at least 5 minutes:

/ = [ (256 - 256 ) × 1000 × 3] (964 × )

A 256 : slope of the initial linear portion of the curve, [unit absorbance/minute] for the Test (with enzyme) and Blank. 

 964: extinction coefficient (L/(mol × cm) of BTEE at 256 nm. 

X: quantity of chymotrypsin in the final reaction mixture (quartz cuvette) [mg] Check that the same activity is obtained for the two tested concentrations.
section_name: Calculations:

section_content: References: Carrière and co-workers 6-8 and Erlanson & Borgström 9 Method: pH-stat titration
section_name: Pancreatic Lipase activity assay (EC 3.1.13)

section_content: Tributyrin + H 2 O pancreatic lipase and cofactor: colipase > butyric acid + sn-2 monobutyrin

The pancreatic lipase activity assays can be conducted using the pH-stat technique using tributyrin as substrate. The free fatty acids released by the lipase are titrated at a constant pH by sodium hydroxide (NaOH, 0. 1N) during the course of the hydrolysis.
section_name: Principle:

section_content: The pancreatic lipase activity must be expressed in international units (U) with 1 U = 1 µmol butyric acid released per minute at 37°C and at pH 8. 0; specific activity of pure Human Pancreatic Lipase is 8,000 U/mg protein on tributyrin 7.
section_name: Unit definition:

section_content: The titration is performed at 37°C and at pH 8. 0, a pH value at which gastric lipase is not active and carboxyl ester lipase is poorly active.
section_name: Conditions:

section_content: Preparation of reagents:
section_name: Protocol

section_content: To measure the trypsin and chymotrypsin activity of the pancreatin (porcine pancreatin (8 x USP specifications, ref P7545

Sigma-Aldrich), the protocols are the same as described here above. Pancreatin is dissolved in 1 mM HCl (pH 3). The dissolution of pancreatin is difficult. After dissolution, keep on mixing during 10 minutes using a magnetic stirrer and then store the pancreatin solution on ice or at refrigerated temperature 4°C. Dilute the enzyme to a concentration ranging between 0. 1 to 1 mg/mL and measure at least 3 different dilutions of the pancreatin in this range. Just before adding the enzyme to the reaction vessel, vortex pancreatin a few seconds. For measuring the lipase activity in pancreatin, it is recommended to dissolve in 150 mM NaCl and adjust pH at 6. 8 (pancreatic lipase is degraded at low pH), and follow the above procedure 10.
section_name: Pancreatin activity

section_content: The concentration of bile salts in the bile (fresh or commercial) is tested using a commercial kit (bile acid kit, ref 1 2212 99 90 313, DiaSys Diagnostic System GmbH, Germany or similar). Follow the supplier's protocol. Prepare and dilute the bile at different concentrations. Check that the concentration of bile salts measured is less than 150 µmol/L in your bile solution, otherwise dilute the bile to be in this range.
section_name: Bile salts assays