Adhesion assay

2.4.1 Mucin

Affinity chromatography experiments were done in batches using 50 mg MUC2-Sepharose bead-slurry in Eppendorf tubes. MUC2 beads were washed with two cycles of 200 μl acetate buffer (0.1 M, pH 3.8, 0.5M NaCl) followed by 200 μl Tris-HCl buffer (0.1 M, pH 8.2, 0.5M NaCl). Beads and buffer were mixed by gentle vortexing followed by a 1 to 2 sec centrifugation with a table top centrifuge (Eppendorf MiniSpin Plus, Eppendorf, North Ryde, Australia). All beads (MUC2 and EtOH-amine) were washed twice with 200 μl PBS to adjust the beads to the condition during adhesion of digest. One ml digest, whey or milk was added to the beads and the suspension was incubated for 30 min at 37°C in a shaking water bath at 110 rpm. After the incubation was over, the supernatant was removed from the beads. To analyse the protein binding a wash cycle as shown in Figure 2.1 was applied. Thereby all removed solutions were collected as they might contain specifically bound proteins. The sequence consisted of 3 x 200 μl PBS, 3 x 200 μl PBS 5.5 (10 ml PBS + 6 ml MilliQ water, pH 5.5 by 1 M HCl), 3 x 200 μl 20% EtOH (in PBS 5.5) and 3 x 200 μl 2.5 M lithium chloride (LiCl (Sigma Aldrich), in PBS 5.5). A potential last wash with 6 M guanidine HCl (in PBS 5.5) was made redundant after deciding not to use affinity chromatography columns and thus no repeated use of the same beads was required. After the wash with PBS, treatment was stopped for an aliquot of the beads (“bead A”). This was used to analyse generally adhering proteins. The leftover beads underwent the wash cycle including 2.5 M LiCl (“bead B”). The beads were mixed with 70 μl 2 x Tris- tricine sample loading buffer (TT-SLB, BioRad) and boiled for 5 min. The removed wash supernatants were collected. The pooled samples were either precipitated or directly mixed 1:1 with 2 x TT-SLB and boiled for 5 min. Prepared beads and supernatants were stored at -20°C until use in SDS-PAGE analysis.

Controls for this assay were undigested whey or milk, MUC2 beads pure and EtOH-amine beads pure, digest, time 0 control (acidified whey + simulated gastric fluid) and pepsin control (pepsin solution + simulated whey + bicarbonate solution + simulated duodenal fluid) were

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Figure 2.1: Outline of the final adhesion assay

The assay was designed to investigate the adhesion of food component of interest to intestinal surface components followed by a sequential wash to remove adhering molecules according to the nature of their binding. Pellet A and pellet B in the figure will be named “bead A” and “bead B” in samples with mucin coated Sepahorse beads, and “pellet A” and “pellet B” in samples containing bacterial cells or IEC.

1. Substrate (binding buffer) and surface component mixed

2. Mix incubated for a set time at 37°C, if possible under agitation

After incubation, supernatant removed

3. Pellet wash with protein free binding buffer Supernatant collected

Pellet A subsampled

4. Pellet wash with PBS 5.5

Supernatant collected

5. Pellet wash with 25% EtOH Supernatant collected

6. Pellet wash with 2.5 M LiCl Supernatant collected Pellet B sampled Wash PBS Pellet A Samples Protocol Wash LiCl Pellet B Wash PBS 5.5 Wash EtOH

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included. Simulated protein-free whey was made up of 8.74 g lactose (M&B Laboratory Chemicals), 0.89 g KCl and 0.33 g CaCl2 (BDH Chemicals Ltd.) in 200 ml MillliQ water.

2.4.2 Bacteria

2.4.2.1 Model 1 for bacterial biofilm: isolated biofilm components

GlcNAc was coated onto Sepharose beads similarly as described for mucin. The only differences were the compositions of coupling buffer (0.1 M NaOH) and storage buffer (0.05 M sodium acetate/acetic acid buffer (pH 4.5)).

2.4.2.2 Model 2 for bacterial biofilm: cell pellets from liquid culture

Bacteria stocks were stored at -80°C in 50% glycerol. In preparation for the experiment, aliquots of the stocks were transferred into 2 ml growth medium and incubated overnight at 37°C with agitation at 180 rpm. The next day, the bacteria were diluted 1:100 into 2 ml fresh medium and incubated under the same conditions. After a further 22 to 24 hr incubation, the cultures were used for the adhesion assay. Preparation consisted of gentle centrifugation (10 min, 1.5 x 103 x g) and 2 x 200 μl PBS-wash of the bacterial cultures to obtain a cell pellet which can then be used for the adhesion assay. The adhesion assay protocol was the same as for MUC2. The bacterial cell pellets were mixed with tagged whey or digest and incubated for 30 min (37°C, 90 rpm, dark).

After incubation, the pellets were washed with 2 x 200 μl PBS; the pellets and PBS were mixed by gentle pipetting followed by a centrifugation (1 min, 7.5 x 10 3 _{rpm) to sediment the bacterial} cells. At this point “pellet A” was sampled. The wash cycle continued with 2x200 μl washes of each of PBS 5.5, 25% EtOH in PBS 5.5 and 2.5 M LiCl in PBS 5.5. The fully washed pellet, “pellet B”, was mixed with 70 μl 2 x TT-SLB, boiled for 5 min and diluted with 140 μl 1x TT- SLB (2 x TT-SLB:MilliQ water / 1:1). The removed wash solutions were collected, and the same solutions were pooled per pellet. The pooled samples were either precipitated or directly diluted 1:1 with 2 x TT-SLB and boiled for 5 min. Prepared pellets and supernatants were stored at -20°C until use for SDS-PAGE analysis.

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2.4.2.3 Whey sediment

To identify the composition of the whey sediment, 500 μl aliquots of whey were centrifuged at 7.5 x 103 rpm for 1 min (as in the adhesion assay) and supernatant was removed from the tube. The sediment was prepared in 20 μl 2 x TT-SLB and run on SDS-PAGE (gel shown in Figure 4.11).

To remove calcium-phosphate from the whey, the whey was incubated at room temperature or at 45°C and the optical density at 600 nm (OD600), as indicator for turbidity, was measured every 10 min for 1 hr. It was determined that a cycle of six 10 min incubations at 45°C without shaking removed most insoluble calcium-phosphate satisfactorily. After each 10 min- incubation, the whey was centrifuged for 1 min at 7.5 x 103 _{rpm. The supernatant was} transferred into a new tube for the next 10 min incubation. The final whey product was free of most calcium-phosphate residue. However, not 100% of the sedimentable material, which also contained low levels of β-LG (compare Figure 4.13), could be removed.

2.4.3 Intestinal epithelial cells

On the day of the experiment (day 16 to 18 post passage), the IEC were washed twice with 500 μl serum free medium. Next, 900 μl serum free medium was transferred to each well and 100 μl Rhd-whey (Figure 2.2) or 100 μl FCS (first sets) or serum free medium (last set) as negative control were added. Cells were then incubated for 15 min, 30 min, 60 min or 120 min in the incubator. After the incubation, the medium was aspirated and the cells were washed twice with 500 μl serum free medium. Half the samples and negative controls were incubated with 100 μl 0.5% dithiothreitol (DTT, ClaBioChem, Merck Millipore, Auckland, New Zealand), a reducing agent that targets disulphide bonds between proteins, in serum free medium in the dark to remove mucin layers by cleaving the “network knots”. This was done for all types of cell cultures (i.e. Caco-2, co-cultures, HT29-MTX), although Caco-2 cells do not secrete mucin [161], to obtain comparable cell fractions for analysis. After 10 min, the cells were rinsed with the medium to collect cleaved mucin. The medium containing mucin (more generally: molecules detached by DTT ) was then transferred into 100 μl 2 x TT-SLB, samples were

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Caco-2 monoculture

Caco-2/HT29-MTX co-culture (90/10)

HT29-MTX monoculture

Tight

monolayer

of

absorptive

enterocyte-like cells

Monolayer

of

90%

absorptive

enterocyte-like cells with 5% mucin

producing

and

5%

non-mucin

producing HT29-MTX cells

Lose monolayer of 50% mucin

producing cells

Caco-2 cells

Non-mucin producing HT29-MTX cells

Mucin

Mucin producing HT29-MTX cells

Rhd-labelled whey

Representative adhesion assay

Incubation of Rhd-labelled whey

with cells in culture, e.g. co-culture

74

labelled “mucin”. This nomenclature was decided upon to clearly differentiate between mucin and cell fraction in the cell culture model, although Caco-2 derived samples were not expected to contain mucin.

The cells were then washed once more with 500 μl serum free medium, treated with 150 μl 2 x TT-SLB and transferred into tubes (“cells”). The other half of samples was directly treated with 150 μl 2 x TT-SLB and transferred into tubes (“whole lysate”). All samples were boiled for 5 min and then stored at -20°C until use.