Novel multiplex PCR-RFLP assay discriminates bovine, porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

ABSTRACT

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.

GRAPHICAL ABSTRACT

KEYWORDS:

• Multiplex PCR-RFLP
• RFLP fingerprints
• halal-authentication
• pharmaceutical products
• universal fish primer
• gelatin

Ethical compliance

This study does not involve any experiments with live animals. Additionally, all animal tissue samples were handled following the institutional and national guidelines for animal care.

Acknowledgments

This work was supported by University of Malaya Research Grant No. FP054-2016 to M.E. Ali. The authors acknowledge the kind gift of wildlife Malaysia and Dewan Bandaraya Kuala Lumpur (DBKL) for monkey, dog and cat meat samples.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by the Institut Pengurusan dan Pemantauan Penyelidikan, Universiti Malaya [FP054-2016 (Dr. Md. Eaqub Ali)].