Lactase

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Lactase (β-d-galactoside galactohydrolase) is a hydrolytic enzyme derived from the mold Aspergillus oryzae. It contains NLT 30,000 USP Lactase Units/g.

[Note—1 USP Lactase Unit is the lactase activity contained in the amount of enzyme that hydrolyzes 1 microequivalent of galactosidic linkage/min at a pH of 4.5 and at 37°, as directed in the Assay for Lactase Activity.]

2 ASSAY

Lactase Activity

Solution A: Dilute 57.5 mL of glacial acetic acid with suficient water to make a 500-mL solution. Transfer 50 mL of the glacial acetic acid solution into a 1000-mL volumetric flask, add 11.3 mL of 4 N sodium hydroxide, and dilute with water to volume. If necessary, adjust with glacial acetic acid solution or 4 N sodium hydroxide to a pH of 4.50 ± 0.05.

Substrate solution: On the day of use, weigh 370.0 mg of o-nitrophenyl-β-d-galactopyranoside, and place in a 100-mL volumetric flask. Add about 50 mL of Solution A, swirl to dissolve, and then dilute with Solution A to volume.

Standard solution: Prepare a 0.4 mg/mL solution of USP Lactase RS in water by allowing the material to stand for 15 min in a small volume of water; swirl gently, and dilute with water to the final concentration. Pipet 3.0 mL of this solution into a 200-mL volumetric flask, and dilute with water to volume.

Sample solution: Prepare 0.4 mg/mL solution of Lactase in water by allowing the material to stand for 15 min in a small volume of water; swirl gently, and dilute with water to the final concentration. Pipet 3.0 mL of this solution into a 200-mL volumetric flask, and dilute with water to volume.

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Mode: Vis

Analytical wavelength: 420 nm

Cell: 1 cm

Analysis

Samples: Standard solution and Sample solution

Pipet 2.0 mL of Substrate solution into 3 separate test tubes labeled "S", "U", and "B". Transfer the tubes to a thermostated water bath maintained at 37.0 ± 0.1°, and incubate for 10 min. Following the incubation, rapidly add 0.5 mL of the Standard solution to tube S, 0.5 mL of the Sample solution to tube U, and 0.5 mL of water to tube B (the reagent blank). Mix each tube on a vortex mixer for 1 s, and immediately return the tubes to the water bath, which has been maintained at 37.0 ± 0.1°. After 15 min of incubation, rapidly add 2.5 mL of a 10% sodium carbonate solution to each test tube to stop the enzyme reaction. Add 20.0 mL of water to each test tube, and mix. Concomitantly determine the absorbances of the 3 solutions.

Calculate the number of USP Lactase Units in the portion of Lactase taken:

Result = [(A_U − A_B)/(A_S− A_B)] × P × (W_S/W_U)

A _U= absorbance of the Sample solution (tube U)

A_B = absorbance of the reagent blank (tube B)

A_S = absorbance of the Standard solution (tube S)

P = potency of USP Lactase RS (USP Lactase Units/g)

W_S = weight of USP Lactase RS in the Standard solution (g)

W_U = weight of Lactase in the Sample solution (g)

Acceptance criteria: NLT 30,000 USP Lactase Units/g

3 IMPURITIES

Change to read:

Arsenic 〈211〉, Procedures, Procedure 1 (CN 1-Jun-2023) : NMT 3 μg/g

Change to read:

Lead 〈251〉, Procedures, Procedure 1 (CN 1-Jun-2023) : NMT 5 μg/g

4 SPECIFIC TESTS

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total aerobic microbial count does not exceed 10³ cfu/g, and the total molds and yeasts count does not exceed 10² cfu/g. It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.

Loss on Drying 〈731〉

Analysis: Dry under vacuum at 60° for 4 h.

Acceptance criteria: NMT 6.0%

5 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers at room temperature.

Labeling: Label it to indicate lactase activity in USP Lactase Units.

USP Reference Standards