Table 3.

Overview of different analytical methods for ALP activity determination in dairy products

Method principle	Wavelength (nm)	Substrate (S)/product (P)	Units definition	Detection limit	RSDR (%)	Scope of usea	Short description	Reference	Remarks
Fluorimetric	Ex 440/Em > 505	S: monophosphoric ester Fluorophos® P: Fluoro‐yellow	U = μmol/min	0.006% raw milk	4.4	Part 1: liquid dairy products from cow, sheep, and goat Part 2: cheese	Measuring fluorescence in the sample with aromatic monophosphoric ester	ISO 11816‐1 | IDF 155‐1:2013 and ISO 11816‐2 | IDF 155‐2:2016 (Rocco, 1990; Shakeel‐ur‐Rehman et al., 2003)	Fluoro‐Test FML 200, Fluorophos method
	Ex 365/Em 450	S: 4‐methyl‐umbelli‐ferone‐phosphate P: 4‐methyl‐umbelliferone	mU = nmol/min	0.006% raw milk	9.5	Fluid dairy products	Assay in a 96‐microwell plate, 4‐MUP as substrate, against a standard curve with 4‐MU of known concentration	Ziobro and McElroy (2013)
			U = μmol/min		4.1	Dairy products (fluid and solid)		ISO/AWI TS 4985|IDF/RM 256 under development
			NR	0.006% raw milk	NR	Purified ALP	Reaction of purified ALP from calf with substrate for 30 min at pH 7.9 or 9.6 at 37°C	Fernley and Walker (1965)
	Ex 405/Em 519	S: trifluoromethyl‐b‐umbelliferone phosphate P: trifluoromethyl‐b‐umbelliferone	Relative fluorescent units	0.04% raw milk	1.5	Liquid and solid samples especially high fat products	Reverse micellar media (microemulsions) used for determination of enzyme activity with the non‐fluorescent substrate.	Fenoll et al. (2002)
Colorimetric	Dependent on substrate	S: phenyl phosphate, phosphate, sodium phenyl phosphate P: phenol	1 μg phenol/min per L	0.1–0.2% raw milk	NR	Whole milk, skim milk, chocolate milk	ALP activity equimolar with release of phosphate from substrate, reaction of phenol with colorimetric compound, Folin–Ciocalteu or 2,6‐dibromoquininechloroimide (BQC)	Shakeel‐ur‐Rehman et al. (2003)	Previous methods: Scharer (1938), ISO 3356:2009, AOAC 972.17
	620 nm	S: phenyl phosphate P: phenol	μg of phenol released/h	NR	NR	Liquid samples	MFO‐3 method: kinetic measurement during 1 h with phenyl phosphate, colour reaction with phenol: CQC (2,6‐dichloroquinone‐4‐chlorimide, incubated for 15 min	MFO‐3 (1981)	Official method in Canada
	NA		1 μg phenol/mL = 500 U/L	6.7 × 10−14 mol/L Phenol = 6.31 × 10−6 μg phenol/L	4.5	Liquid milk	Electrochemical determination, Graphite–Teflon composite tyrosinase biosensor monitors by ALP produced phenol through reduction of o‐quinone	Serra et al. (2005)
	Blue – green	S: p‐Nitrophenol phosphate P: phenol	pos/neg	NR	qualitative > 0.5 units/L	Liquid milk from buffalo, cow and goat	Visual inspection Rapid test with using this principle: on strip immobilised p‐nitrophenyl phosphate reacts with ALP and produces p‐nitrophenol that reacts with a chromogen producing a colour change	Sharma et al. (2003)	IDF 82A, 1987 and available as rapid test with Dry‐reagent strips
	NA	S: 5‐bromo‐4‐hloro 3‐indolyl phosphate (BCIP) P: phenol	pos/neg	% of raw milk: n.d. (0.87 U/mL based on SD)	< 5.1	Milk	Biosensor, miniaturised: digital image colorimetry with smartphone. ALP antibody immobilised on paper, substrate BCIP	Mahato and Chandra (2019)
Chemiluminescent	540 nm	S: 3‐(2′‐spiroadamantanane)‐4‐methoxy‐4(3″‐phosphate phenyl‐1,2 dioxetane disodium salt (Charm reagent®) AP P: adamantly1,2‐dioxetan	U = μmol/min	0.05–0.2% raw milk	7.5	Liquid milk from cow, goat and sheep, flavoured drink and cream	Photo‐activation of hydrolysed product (chemiluminescent), kinetic stop reaction, Charm ALP‐Paslite, Chemi‐F‐AP	Albillos et al. (2011); ISO 22160:2007; Salter and Fitchen (2006)	Charm Test, ISO‐22160:2007, Rapid test: Chemi‐F‐AP

ALP: alkaline phosphatase; Ex: Excitation; Em: Emission; RSDR: relative standard deviation of reproducibility; NA: not applicable; NR: not reported; neg: negative; pos: positive; SD: Standard deviation.

^aWhen species were indicated it was added to the table, otherwise there was no indication in the reference.