Development and validation of an enzymatic assay for the measurement of lactose in lowlactose and lactose-free products

Transcription

1 Development and validation of an enzymatic assay for the measurement of lactose in lowlactose and lactose-free products Barry McCleary, David Mangan, Helena Culleton, Claudio Cornaggia, Ruth Ivory, Vincent McKie, Elaine Delaney and Tadas Kargelis

2 Lactose what is it? D-Lactose Present in human milk at ~7% w/v and bovine milk at ~5% w/v b-galactosidase (Lactase) + D-Galactose Incorporated into glycolipids, glycoproteins or converted to D-glucose in the liver D-Glucose Metabolised to produce energy

3 Lactose intolerance what is it? Congenital lactose intolerance Life-long condition Single autosomal recessive disorder Extremely rare Primary lactase non-persistence Lactase activity declines after weaning Affects ~70% of population Secondary lactase non-persistence Occurs in lactase persistent individuals Caused by gastrointestinal illness that damages the brush border of the small intestine Potentially reversible M.C. Lomer et al. Aliment Pharmacol Ther. 2008, 27(2),

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5 Regulations on low-lactose/lactose-free products Territory Low-lactose Lactose-free EU (EFSA) Limit undefined 10 mg/100 kcal Germany Slovenia Limit undefined 100mg/100g Hungary Denmark Estonia Finland 1g/100g 10mg/100g Norway Sweden Ireland 1g/100g No detectable lactose or galactose United States Limit undefined Limit undefined Canada 25% reduction c/w standard version No detectable lactose Australia New Zealand 2 g lactose/100g food No detectable lactose China 2g/100g 0.5 g/100g Singapore 5g/100g No detectable lactose Sources: EFSA, FDA, Canadian Food Inspection Agency, FSANZ (Food Standards Australia New Zealand), CFDA (China Food and Drug Administration), AVA (Agri-food and Veterinary Authority of Singapore)

6 Instrument based lactose methods with varying sensitivity levels Low sensitivity IR spectroscopy (AOAC method ) Polarimetry (AOAC method ) Gravimetry (AOAC method ) Medium sensitivity HPLC employing refractive index (RI) detection J.L. Chavez-Servin et al. J Chromatogr A (2), HPLC employing evaporative light scattering (ELSD) detection R. Schuster-Wolff-Bühring et al. Dairy Sci Technol (1): High sensitivity and resolution HPAEC-PAD chromatography W.B. van Scheppingen et al. J Chromatogr B (Supplement C), L. Monti et al. Food Chem (Supplement C),

7 Instrument based lactose methods with varying sensitivity levels Low sensitivity IR spectroscopy (AOAC method ) Polarimetry (AOAC method ) Gravimetry (AOAC method ) Medium sensitivity HPLC employing refractive index (RI) detection J.L. Chavez-Servin et al. J Chromatogr A (2), HPLC employing evaporative light scattering (ELSD) detection R. Schuster-Wolff-Bühring et al. Dairy Sci Technol (1): High sensitivity and resolution HPAEC-PAD chromatography W.B. van Scheppingen et al. J Chromatogr B (Supplement C), L. Monti et al. Food Chem (Supplement C), BUT Significant capital investment required to purchase equipment Specific analytical expertise required to operate Long duration sample run times (>1 hour) results in low throughput

8 Enzymatic lactose determination general principles Residual lactose b-galactosidase Steps in enzymatic measurement of lactose 1) Measure free glucose or galactose in the sample A1 2) Hydrolyse lactose present with β-galactosidase 3) Measure free glucose or galactose released from lactose hydrolysis A2 4) Calculate lactose content by subtracting A1 from A2

9 Lactose determination challenges for enzymatic methods MAJOR PATHWAY Glucose Galactose Hydrolysis Large quantities Manufacture of lactose free products Lactose naturally occurring in dairy products β-galactosidase treatment Remaining lactose, plus: MINOR PATHWAY Transglycosylation Allolactose (β-1,6-lactose) β-1,3-lactose β-1,2-lactose Galacto-oligosaccharides (GOS) Trace quantities

10 Lactose determination challenges for enzymatic methods 1. High background signal arising from either free glucose or galactose In untreated low lactose samples, the difference between A1 and A2 is miniscule in comparison to A1 which leads to large errors A2-A1 The difference between A1 and A2 is now very large in comparison to A1 which greatly reduces errors A1 A2 A1' A2' A2 - A1

11 Lactose determination challenges for enzymatic methods 1. Sensitivity. A) High background signal galactose determination McCleary, B.V. and Charnock S. Assay for determination of free D-galactose and/or L-arabinose. EP (B1).

12 Lactose determination challenges for enzymatic methods 1. Sensitivity. B) High background signal glucose determination Glucose + O 2 + H 2 O Glucose oxidase Catalase Gluconolactone + H 2 O 2 Glucose oxidase/catalase pre-treatment

13 Lactose determination challenges for enzymatic methods 1. Sensitivity. B) High background signal glucose determination Glucose + O 2 + H 2 O Glucose oxidase Catalase Gluconolactone + H 2 O 2 Glucose oxidase/catalase pre-treatment

14 Lactose determination challenges for enzymatic methods MAJOR PATHWAY Glucose Galactose Hydrolysis Large quantities Manufacture of lactose free products Lactose naturally occurring in dairy products β-galactosidase treatment MINOR PATHWAY Transglycosylation Allolactose (β-1,6-lactose) β-1,3-lactose β-1,2-lactose Galacto-oligosaccharides (GOS) Trace quantities

15 Lactose determination challenges for enzymatic methods 2. Selectivity. A) β-galactosidase hydrolysis of lactose versus lactose analogues

16 Lactose determination challenges for enzymatic methods 2. Selectivity. A) β-galactosidase hydrolysis of lactose versus lactose analogues

17 Lactose determination challenges for enzymatic methods 2. Selectivity. B) Creep calculation to account for partial hydrolysis of allolactose

18 Lactose determination challenges for enzymatic methods 2. Selectivity. B) Creep calculation to account for partial hydrolysis of allolactose Analysis of 25 mg lactose in the presence of varying amounts of allolactose

19 Lactose determination challenges for enzymatic methods 2. Selectivity. B) Creep calculation to account for partial hydrolysis of allolactose

20 Lactose determination challenges for enzymatic methods 3. Assay format: non-sequential versus sequential Existing assay formats operate in a non-sequential manner with analysis of free glucose (A1) and glucose released from lactose (A2) performed in separate cuvettes Test sample Glucose detection Read A1 β-galactosidase plus glucose detection Read A2

21 Lactose determination challenges for enzymatic methods 3. Assay format: non-sequential versus sequential Existing assay formats operate in a non-sequential manner with analysis of free glucose (A1) and glucose released from lactose (A2) performed in separate cuvettes K-LOLAC assay format employs the key enzyme, β-galactosidase, at a much higher activity level which allows determination of A1 and A2 in a single cuvette. Test sample Test sample Glucose detection Read A1 β-galactosidase plus glucose detection Read A2 Glucose detection: Read A1 then, add β-galactosidase: Read A2 A sequential assay format results in reduced analyst handling which increases accuracy while decreasing associated reagent and resource costs

22 K-LOLAC novel assay procedure for lactose measurement 1. Sensitivity: A) Removal of glucose using glucose oxidase/catalase B) Improved glucose detection biochemical pathway using 6-phosphogluconate DH 2. Selectivity: A) Selectivity of MZ104 β-galactosidase for lactose in the presence of allolactose B) Automated creep calculator allows accounting for and removal of absorbance contribution arising from allolactose 3. Assay format: Introduction of sequential assay format to improve accuracy and reduce analyst input

23 K-LOLAC novel assay procedure for lactose measurement

24 K-LOLAC Linearity

25 K-LOLAC Intermediate precision Number of analyses µg Lactose in cuvette Ref Material (g L -1 ) Mean Value (g L -1 ) Standard Deviation %CV LOD = 0.15 mg/l LOQ = 0.25 mg/l 0.79 mg/l for liquids and 0.13 mg/100 g for solids 2.65 mg/l for liquids and 0.44 mg/100 g for solids

26 K-LOLAC real sample analysis

27 K-LOLAC real sample analysis Galactose can inhibit MZ104 β-galactosidase Overcome by 2x increase in the β-galactosidase Units employed

28 K-LOLAC real sample analysis Sample Physical form Lactose in sample ( g L -1 ) Recovery of cuvette spike (%) Recovery of sample spike (%) Adult nutritional shake Liquid Kefir Liquid Low-lactose milk Liquid Low-lactose milk Liquid Low-lactose UHT milk Liquid Rice milk Liquid Soya milk Liquid Brie cheese Solid Cheddar cheese Solid Coffee creamer Solid Cottage cheese Solid Natural live yoghurt Solid Parmesan cheese Solid < Processed cheese slices Solid Ricotta cheese Solid Low-lactose cookies Solid Low-lactose nachos Solid Low-lactose pudding Solid Low-lactose tart Solid

29 K-LOLAC baby formula sample analysis Lactose-free infant formula sample A Product label lactose content (mg per 100 g) Not stated B 50 C D Not stated Not stated E <42 F <52 Analysis method Lactose (mg per 100 g) Lactose (mg per 100 kcal) Spike recovery (%) MZ EcLacZ HPAEC-PAD MZ EcLacZ HPAEC-PAD MZ EcLacZ HPAEC-PAD MZ EcLacZ HPAEC-PAD <1 N/A 98.0 MZ EcLacZ HPAEC-PAD MZ EcLacZ HPAEC-PAD

30 Conclusions Development of the first enzymatic method suitable for the measurement of residual lactose in low-lactose or lactose-free products Highly sensitive, selective and accurate Fully characterised in terms of: Linearity ( mg per assay) Intermediate precision (CV ~3%) LOD (0.79 mg/l for liquids and 0.13 mg/100 g for solids) LOQ (2.65 mg/l for liquids and 0.44 mg/100 g for solids) Demonstrated for a range of low-lactose and lactose-free products