Test solutions

2.3.1 Whey and whey preparations

2.3.1.1 Acid whey

Raw milk was sourced from the local AgResearch farm (Tokanui farm, AgResearch, New Zealand). Whey was produced by acidic precipitation of raw milk. Agitated raw milk was heated to 40°C in a water bath. The milk was acidified with 2 M HCl, decreasing the pH-value to 4.7. Acidification was continuously controlled with a pH-meter to avoid a drop in pH-value under the desired value. After reaching the final pH-value, stirring was stopped and the curd was allowed to set for 1 hr in the water bath. Before filtering the settled curd and whey through a cheese cloth, the pH-value was checked and adjusted to 4.7 if necessary. The separated whey was collected and centrifuged for 30 min at 3 x 103 x g. The clarified fractions were pooled and the pH-value adjusted to 6.8 by adding 1 M NaOH. The whey was heated to 40°C in a water

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bath and left without stirring for 30 min. To remove any calcium-phosphate precipitate, the whey was again centrifuged for 30 min at 3 x 103 _{x g. The clear fractions were pooled, aliquoted} into Falcon tubes (LabServ, Thermo Fisher Scientific, Auckland, New Zealand) and stored at - 80°C until use.

2.3.1.2 Whey using centrifugation

Skim milk was centrifuged at 105 x g for 60 min at 6°C (Sorvall® Discovery 90SE, Thermo Fisher Scientific, Auckland, New Zealand). The clear whey was removed from the tubes, pooled and aliquoted into Falcon tubes. These were stored at -80°C until use.

From here, “whey” is referred to whey produced by acidification and “whey by centrifugation” refers to whey produced by centrifugation.

2.3.1.3 Protein quantification by Bradford assay

For the Bradford assay, a 7-point BSA-standard (STD) dilution series was prepared, ranging from 0 to 18 μg BSA in 100 μl MilliQ water. Whey was used pure or as 1:10 dilution in MilliQ water in 100 μl aliquots. 900 μl Bradford solution (BioRad) was added to STDs and samples, mixed and allowed to react for 10 min at room temperature. The STDs were measured at 595 nm on a photometer (UV-160A, Shimadzu, Auckland, New Zealand) to generate a STD curve. Next the samples were measured and their protein content calculated. Bradford solution was made from 20x stock solution and stored in the dark at room temperature.

2.3.1.4 Protein quantification with DirectDetect

DirectDetect (Merck Millipore, Auckland, New Zealand) is an alternative method to measure the protein content. It measures the amide bonds and is therefore less susceptible to differences in amino acid sequence or dye binding properties between the calibration protein – routinely BSA – and the sample protein (mix) than colorimetric methods like Bradford. 2 μl samples and one blank (buffer only) were pipetted onto the measurement cards which were then ready for quantification [464].

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Protein quantification by Bradford and DirectDetect were compared and are discussed in Section 3.2.1.1.

2.3.1.5 Partially digested whey

To generate a range of whey protein samples, digested to various degrees as needed for the adhesion assay, whey was partially digested with pepsin and adjusted to duodenal conditions (ionic strength and pH-value). The assay was optimised from the US Pharmacopeia standard gastric digestion assay for protein [465]. Testing adhesion properties of a partial in vitro gastric

digest was initially considered as proteins are hydrolysed in the stomach before reaching the small intestine and thus modified proteins are expected to be available for adhesion. However, this approach was dropped quickly as comprehsive analysis of digestion products was not possible.

Pepsin solution at physiological concentrations was made fresh for every digest; pepsin from porcine gastric mucosa (Sigma Aldrich) was dissolved in simulated gastric fluid (150 mM NaCl, 10 mM HCl, pH 2.0). This was done in two steps: first 45 mg pepsin was dissolved in 20 ml simulated gastric fluid in a shaking water bath (WiseBath®, Wisd. Laboratory Instruments, Witeg Labortechnik GmbH, Wertheim, Germany) at 37°C and 90 rpm. After 30 min 1.86 ml of this solution was added to 18.14 ml simulated gastric fluid and incubated for another 30 min under the same conditions, resulting in 20 ml digest buffer with a final concentration of 0.21 mg.ml-1 pepsin. About 15 ml whey was acidified with 1 M HCl to pH 2 and warmed to 37°C in the water bath. To start the digest, 10 ml acidified whey and 5.77 ml digest buffer were mixed; the protein:enzyme ratio was 20:1. After 1 min, the digestion was stopped by mixing 6.86 ml digest, 0.85 ml bicarbonate solution (250 mM NaHCO3 (Sigma Aldrich)), 34.0 μl Pepstatin A (Sigma Aldrich) and 2.29 ml simulated duodenal fluid (60.1 g.L-1 NaCl, 4.6 g.L-1 _{KCl, 1.7 g.L}-1 _CaCl

2·2H2O (BDH AnalaR), 0.1 M Tris (Fisher Scientific), pH 7.5). The digest was used immediately for the adhesion assay.

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2.3.2 Protein labelling

Throughout the project, different fluorescent tags were used: DyLight594 (DL594, Thermo Scientific), Rhodamine (Rhd, Thermo Fisher Scientific) and FITC (Sigma Aldrich). Rhd and DL594 came as N-hydroxysuccinimide-ester (NHS-ester) and were simply added to the (dialysed) whey and incubated at room temperature. FITC was used for flow cytometry experiments because the instrument was not able to detect the other tags.

2.3.2.1 DyLight594 labelling of whey

DL594 tagging of proteins was done according to manufacturer’s instructions. In brief, whey or digest were dialysed against PBS overnight with three buffer exchanges through a 3.5 kDa cut- off membrane (Spectrum Laboratories Inc., Auckland, New Zealand). Then next morning, whey or digest was mixed with fluorophore (1 mg.ml-1 _{in PBS) at 15 μg label per 1 mg protein. The} mix was allowed to react for 1 hr in the dark before using it in the adhesion assay.

2.3.2.2 Rhodamine labelling

Rhd-NHS was coupled to protein according to the manufacturer’s instructions. Some grains of Rhd were dissolved in 100 μl dimethyl sulfoxide (DMSO, Sigma Aldrich) and this solution was mixed with whey or digest. After incubating for 1 hr in the dark, an equal volume of 1 M Tris, pH 7 was added to block yet unbound NHS-groups. After incubation in the dark for 30 min, it was ready for immediate use.

The Rhd was kept as a powder to avoid premature degradation. Thus fresh Rhd was made up for each labelling. As the amount of Rhd was too small for precise measurement, each experiment can only be compared with itself. For comparison between different experiment days, all values were converted into percentages of whey, which was loaded at 5 μl as reference onto each gel.

2.3.2.3 FITC labelling of proteins for Flow Cytometry

Isolated proteins were dissolved in 0.1 M sodium-bicarbonate, pH 9.0, at concentrations from 3 mg.ml-1 to 25 mg.ml-1. FITC was made up at 5 mg.ml-1 in the same buffer. FITC-label was added in 10-fold molar excess to protein under shaking. The mix was incubated for 2 hr in the

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dark. After this, 1.5 M hydroxylamine-HCl (Sigma Aldrich), pH 8.5 was added to block yet unbound FITC (same volume as FITC). To remove unused label from labelled protein, the mix was cleaned over a 10DG desalting column (BioRad; prepacked with Bio-Gel®P-6DG gel). The column was equilibrated with PBS and used according to manufacturer’s instructions. However, due to the high protein content, for some proteins a pre-clean with Sephadex G10 (Sigma Aldrich) was necessary. This medium was hydrated in MilliQ water and the slurry transferred into a column. After equilibrating the column with PBS, the sample was loaded. To remove all sample from the column, which binds free label, the column was centrifuged at 2 x 103 x g for 2 min. After cleaning, the FITC-labelled protein as aliquoted and stored at -20°C in the dark until use.

The degree of FITC-labelling was calculated by comparing the amount of FITC with the amount of protein in solution. FITC was measured with a plate reader (BioTek, Winooski, USA) against a FITC-STD curve. Protein was measured with DirectDetect. Both values were calculated into moles, allowing the determination of mol FITC.mol-1 _protein.

2.3.3 Milk

Skim milk was made by centrifugation of raw milk at 2.5 x 103 _{x g and 4°C for 10 min. A} commercial product (Meadow Fresh, Goodman Fielder, Auckland, New Zealand) was also used.

2.3.3.1 Digested skim milk

Digestion of skim milk was performed as for the whey digestion. The volume ratio of simulated gastric fluid:milk had to be altered due to the higher protein content in skim milk; 15 ml skim milk were mixed with 18.03 ml pepsin solution. The digestion time was pre-determined from a digestion time course. It was set at 30 min (compare Section 2.2.1.3). To stop the digestion, 1.7 ml bicarbonate solution, 68.0 μl Pepstatin A and 4.58 ml simulated duodenal fluid were added to 13.72 ml digest. The digest was used immediately for the adhesion assay.

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