3. Experimental part – Part I
3.17 In vitro gastrointestinal digestion of purified protein
• Egg lecithin (10 mg, Grade 1) (Lipid Products, Surrey England)
• Sodium chloride (NaCl) (Sigma-Aldrich, USA)
• Chloridric acid (HCl) (1.0 M solution in Milli Q H2O) (Sigma-Aldrich, USA)
• Sodium hydroxide (NaOH) (1 M, 0.1 M and 0.01 M solutions in Milli Q H2O) (Sigma-Aldrich, USA)
• Bis-Tris base (Sigma-Aldrich, USA)
• Calcium Chloride (CaCl2) (1 M solution in Milli Q H2O) (Sigma-Aldrich, USA)
• Dichloromethane (CH2Cl2) (Sigma-Aldrich, USA)
• Ammonium bicarbonate (NH4HCO3) (Sigma-Aldrich, USA)
• Ethanol (CH3CH2OH) (Sigma-Aldrich, USA)
• Methanol (CH3OH) (Sigma-Aldrich, USA)
• Sodium taurocholate NaTC (C26H44NO7S•Na) (2.386 mg/mL in CH3OH) (Sigma-Aldrich, USA)
• Sodium glycodeoxycholate NaGDOC (C26H42NNaO5) (2.094 mg/mL in CH3OH) (Sigma-Aldrich, USA)
• Pepsin from porcine gastric mucosa, 3300 u/mg (Sigma-Aldrich, USA)
• Trypsin from porcine pancreas, 13500 u/mg (Sigma-Aldrich, USA)
• α-Chymotrypsin from bovine pancreas, 40 u/mg (Sigma-Aldrich, USA)
• Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, USA)
• Milli Q H2O obtained with Barnstead Nanopure Diamond Uv Toc D11951 (Thermo Scientific)
• N2 flux
• Ar flux
3.17.2 Instrumentation
• Digital scale U4600P Universal (Sartorius, Gemany)
• Digital scale 1602 MP Analytical (Sartorius, Gemany)
• CD 720 pH meter (WPA ltd, UK)
• S2896 B & T Flatspin 12V magnetic stirrer
• Millex Filter Units 0.22 µm (Millipore, USA)
• 10 mL disposable syringes
• Vacuum oven OVL-570-010J (Weiss Gallenkamp, UK)
• Digital Sonifier 250 (Branson Ultrasonics Corporation, USA)
• Excella™ E24 benchtop incubator shaker (New Brunswick Scientific, USA)
• Analytical Balance ME-30 (Mettler Electronics Corp, USA)
• Rotary Evaporator R201B-III (Sunwain Co., Ltd, China)
• TotalLab TL120 - 1D gel image analysis software (Shimadzu Ltd, UK)
98 3.17.3 Procedure
i. Digestion solutions preparation
• Egg lecithin stock solution
A vial containing 10 mg of egg lecithin was poured in 50 mL round-bottom flask, previously cleaned with ethanol and dried by N2 flux. The content was made up to 10 mL with CH2Cl2. Once prepared, it was flushed with Ar flux and stored at -20 °C.
• Simulated Gastric Fluid (SGF)
8.77g of NaCl were dissolved in 900 mL of Milli Q H2O. The pH was adjusted to 2.5 using 1.0 M HCl and the volume was brought to 1000 mL. The solution was filtered using a 0.22-µm filter.
• Simulated Duodenal Fluid (SDF)
8.77g of NaCl were dissolved in 900 mL of Milli Q H2O. The pH was adjusted to 6.5 using 0.01 M NaOH and the volume was brought to 1000 mL. The solution was filtered using a 0.22-µm filter.
• Simulated Duodenal Fluid (SDF) 15x
0.785 g of Bis-Tris base were dissolved in 15 mL of SDF. 187.5 µL of 1 M CaCl2 were added to give 7.5 mM in 25 mL. The pH was brought to 6.5 using 1.0 M HCl and more SDF was added up to 25 mL. The solution was filtered using a 0.22-µm filter and stored at 4 °C.
• 0.5 M ammonium bicarbonate
3.95 g of NH4HCO3 were dissolved in 100 mL of Milli Q H2O and filtered through a 0.22-µm filter.
• Egg lecithin for gastric digestion phase
91.4 µL of egg lecithin stock solution were placed in a 50 mL round-bottom flask. 1 mL of dichloromethane was added and the solution was allowed to dry at the rotary evaporator. After being flushed 4 times with air and three times with Ar, the lecithin was allowed to dry in vacuum oven overnight.
• Egg lecithin for duodenal digestion phase
1 mL of each bile salts solution (NaTC and NaGDOC) were placed into a 20 mL round-bottom flask and 30.8 µL of egg lecithin stock solution were added. The solution was evaporated at the rotary evaporator and, after being flushed 4 times with air and three times with Ar flux, it was dried in vacuum oven overnight.
• Pepsin stock solution preparation
Pepsin solution was prepared in order to be 6628 u/mL, that is 2.008 mg/mL when using 3300 u/mg stock bottle. At least 10-15 mg of pepsin were weighed and made up to 2.008 mg/mL with cold SGF pH 2.5, dissolved by stirring and added to digestion as soon as possible.
• α-Chymotrypsin stock solution preparation
Chymotrypsin solution was prepared in order to be 38.61 u/mL, that is 0.97 mg/mL when using 40 u/mg stock bottle. At least 10-15 mg of chymotrypsin were weighed and made up to 0.978 mg/mL with cold SGF pH 2.5, dissolved by stirring and added to digestion as soon as possible.
• Trypsin stock solution preparation
Trypsin solution was prepared in order to be 3330 u/mL, that is 0.247 mg/mL when using 13500 u/mg stock bottle. At least 10-15 mg of trypsin were weighed and made
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up to 0.247 mg/mL with cold SGF pH 2.5, dissolved by stirring and added to digestion as soon as possible.
ii. Gastric digestion phase
The egg lecithin prepared for gastric digestion phase was re-suspended in 10 mL of pre-warmed SGF pH 2.5 for 20’ at 37 °C in incubator with 3-4 glass beads as dispersing vehicle. Then the suspension was sonicated until it looked pretty clear, using the following settings:
0.9’’ on – 0.1’’ off, for 2’, 40% power, repeated 3 times.
2 mg of purified protein were weighed in a 7 mL Sterilin tube with a magnetic flea; 1 mL of SGF pH 2.5 and 600 µL of egg lecithin for gastric digestion were added. After a brief stir, the pH of the solution was adjusted to 2.5 with 1.0 M HCl and its volume was noted. In order to make up 2 mL, a suitable volume of SGF pH 2.5 was added. 50 µL of sample were taken and added to a 0.5 mL eppendorf tube containing 10 µL of 0.5 M NH4HCO3 (time 0 of gastric digestion).
50 µL of pepsin stock solution were added to digestion mix and the sample was placed in incubator at 37 °C for 1 h in agitation (120 r.p.m.).
At the end of gastric phase, 50 µL of digestum were collected and transferred to a 0.5 mL eppendorf tube containing 10 µL of 0.5 M NH4HCO3 (time 60’). The pH of the remaining solution was adjust to 7.5 with 1 M and 0.1 M NaOH in order to stop the digestion by arresting pepsin activity.
iii. Duodenal digestion phase
1.08 mL of gastric digesta and 80 µL of SDF 15x were added to the lecithin/bile salt mix in the 20 mL round-bottomed flask, flushing with Ar and mix in incubator for 5’.
1.06 mL of the emulsion were taken and added to 7 mL Sterilin tube with small magnetic flea; its pH was adjusted to 6.5 with 1.0 M HCl or NaOH as necessary by adding 1 µL at time using P20 pipette. In order to make up 2 mL, a suitable volume of SDF pH 6.5 was added. After the stock solution of enzymes were prepared, 10 µL of both chymotrypsin and trypsin stock solution were added to the digestion mix. 50 µL of digesta were collected immediately after the addition of the enzymes (time o of duodenal digestion) and placed into 0.5 mL eppendorf tube containing 4 µL of 0.1 M PMSF, a proteinases inhibitor to halt the activity of the enzymes. The duodenal digestion mix was placed in incubator at 37 °C in agitation (120 r.p.m.) and several sampling (50 µL each) were taken after 1, 2, 5, 10, 15, 30, 60 and 120’ of incubation and transferred to 0.5 mL eppendorf tube containing 4 µL of 0.1 M PMSF. Remaining digestum was placed into a 1.5 mL eppendorf tube containing 40 µL of 0.1 M of PMSF solution.
All the samples obtained after the digestion process were analyzed by SDS PAGE under both denaturing and native conditions, as described in paragraph 3.6, and the profile and the relative quantification of protein and peptides were outlined by TotalLab TL120 software.
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