Table 1 Experimental design for assay development

i

Validate linearity of the assay signal

7.75 mM p-nitrophenyl-β-O-d-glucosidase + 0.0025 units of almond β-glucosidase, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min; no α-cyclodextrin

Absorbance measured every minute for 10 min after the addition of almond β-glucosidase

Linearity of the assay is confirmed

ii

Assess interference from saliva

See (i) above

Absorbance values of buffer compared with and without 25 μL heat-inactivated^a human saliva

Heat-inactivated saliva does not interfere with the matrix

iii

Determine absorbance maxima of p-nitrophenyl

See (i) above

Assay measured at 400 and 405 nm

400 nm selected

iv

Determine substrate saturation concentration

Varying substrate concentrations + 0.0025 units of almond β-glucosidase, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min

Substrate concentration varied between 0 and 10 mM

7.75 mM selected

v

Determine maximum saliva volume

7.75 mM p-nitrophenyl-β-O-d-glucosidase + 0.0025 units of almond β-glucosidase + varying volumes of heat-inactivated saliva, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min

Different amounts of heat-inactivated saliva evaluated: 25, 50, 100, 150 and 200 μL

200 μL selected

vi

Evaluate efficacy of α-cyclodextrin

7.75 mM p-nitrophenyl-β-O-d-glucosidase + 0.0025 units of almond β-glucosidase + 200 μL of heat-inactivated saliva, corrected to final volume of 2 mL with 100 mM phosphate buffer (pH 6.5); wavelength 400 nm over 10 min

Assays performed with and without 6 mM α-cyclodextrin

Inclusion of 6 mM α-cyclodextrin in assay