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The outline method given above gives the general approach that should be used in terms of enzymes, their activities, ionic composition and endogenous surfactants. However, there are some practicalities that need to be taken into account when executing the method. For example the simulated digestion fluids (SSF, SGF and SIF) are made up using the electrolyte stock solutions given in Table 2.3, enzymes, bile, CaCl2 and water. The volumes are calculated for a final volume of 500 mL for each simulated fluid. However, we recommend making up the stock solution with distilled water to 400 mL, i.e. 1.25 times concentrated, for storage at −20 °C. The addition of enzymes, bile, Ca2+ solution etc. and water will result in the correct electrolyte concentration in the final digestion mixture. CaCl2 is not added to the electrolyte stock solutions as precipitation may occur. Instead, it is added to the final mixture of simulated digestion fluid and food.


The way that sampling should be done depends on the nature of the study and should be carefully considered for each study. For example, it may be advisable to have individual sample tubes for each time point rather than withdrawing samples from the reaction vessel. Also, it may be important to sample at multiple time points through both gastric and intestinal phases or it may only be necessary at the end of digestion. Regardless of such questions, the way in which the reactions are stopped will depend on what the samples will be subsequently required for. The following are some recommendations to inhibit further enzyme action in the digesta samples:

• Snap freezing of samples is recommended in liquid nitrogen immediately after the reaction for further analysis. It should be born in mind that enzymes will continue to act, even in frozen samples albeit slowly. Therefore the colder the sample is stored the better.

• If samples are sent to other labs, i.e. by courier or by post, the digestion should be stopped completely and for this, the following procedures are recommended:

– Neutralize the pH in the gastric phase by adding 0.5 M sodium bicarbonate. This will inactivate the pepsin before snap-freezing in liquid nitrogen and subsequent storage and/or freeze drying.

– Addition of protease inhibitor (e.g. 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride [AEBSF], Roche or similar), snap freezing in liquid nitrogen and subsequent freeze drying of samples.

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