Monitoring Indian "Superfood" Moringa oleifera Lam. - species-specific PCR-fingerprint-based authentication for more consumer safety

Abstract

Moringa oleifera Lam. has become one of the major new superfoods commonly available in the aisles of bio-shops and health-food sections in supermarkets of North America and Europe. While most of these products appear under the generic and scientifically inconclusive term "Moringa", the European Union, so far, has allowed commercialisation for the use in food and feed for M. oleifera only. M. oleifera is indigenous to India and South Asia, but large-scale cultivation of this species has spread to the tropical regions on all continents, with a strong focus on Africa, leading to a high risk of admixture with species like M. stenopetala (Baker f.) Cufod. that is native to Africa. In the present study, we have characterised six species of Moringa in order to develop a simple and robust authentication method for commercial products. While the plants can be discriminated based on the pinnation of the leaves, this does not work for processed samples. As alternative, we use the plastidic markers psbA-trnH igs and ycf1b to discern different species of Moringa and develop a diagnostic duplex-PCR that clearly differentiates M. oleifera from other Moringa species. This DNA-based diagnostic assay that does not rely on sequencing was validated with commercial products of "Moringa" (including teas, powders, or capsules). Our method provides a robust assay to detect adulterations, which are economically profitable for costly superfood products such as "Moringa".

Fig. 1. Comparison of leaf appearance of Moringa species of our study.

Images of single leaflets (inset) were recorded using a stereomicroscope at a magnification of 6.3x (scale bar in inset is 2 mm). (*) received as M. ovalifolia, by re-evaluation determined as M. oleifera. A Moringa oleifera, (B) M. ovalifolia (*), (C) M. peregrina, (D) M. stenopetala, (E) M. drouhardii and (F) M. hildebrandtii.

Fig. 2. Cells with calcium oxalate glands in the spongy parenchyma of Moringa species.

Calcium oxalate crystals were visualised in the spongy parenchyma layers of the leaf using polarised light. (scale bar 50 μm). (*)received as M. ovalifolia, by reevaluation determined as M. oleifera. A Moringa oleifera, (B) M. ovalifolia(*), (C) M. peregrina, (D) M. stenopetala, (E) M. drouhardii and (F) M. hildebrandtii.

Fig. 3. Macroscopic analysis of commercial products containing Moringa.

Commercial products come in different stages of processing, as complete leafs (A, B), as tea with Moringa leafs (black square) among other plants (C) and as powder (D). Scale bar is 2 mm.

Fig. 4. Neighbour joining phylogenetic tree based on the plastidic ycf1b marker.

The reference plants are represented as coloured squares, with red for Indian M. oleifera, yellow for African Moringa species (including M. hildebrandtii, M. stenopetala and M. drouhardii) and orange for M. peregrina individuals. Commercial products are displayed with a grey square. The outgroups Carica papaya and C. pentagona are represented as black squares. The internal ID of the Botanical Garden of the KIT (see also Table 1) for the ycf1b fragments are given next to the species name. The numbers on the branches indicate the reliability of the clusters by means of 1000 bootstrap replications. The geographic regions are not drawn by scale. (*) received as M. ovalifolia, by re-evaluation determined as M. oleifera.

Fig. 5. ycf1b-based ARMS diagnosis for differentiating between Moringa oleifera and other Moringa species.

The commercial products and the reference plants are highlighted in different colours, according to the phylogenetic tree. A Schematic illustration of the ycf1 region and orientation of primers used in this study. The decisive nucleotide substitutions were detected in the ycf1b region of Moringa. Sequence cutouts of different Moringa species and the primer sequences are highlighted. B Duplex-PCR with ycf1b fw / rv and the additional diagnostic ycf1b_Mo_ARMS_rv primer. All samples display a ycf1b control band with a fragment size of around 950 basepairs. An additional diagnostic ARMS band with a fragment size of 600 basepairs is clearly visible in M. oleifera reference plants (labelled red) and the commercial Moringa products (labelled grey). This specific M. oleifera band is completely absent in African M. stenopetala, M. hildebrandtii and M. drouhardii (labelled yellow) and Arabic M. peregrina (labelled orange). C Duplex-PCR with ycf1b fw / rv and the additional diagnostic ycf1b_NoMo_ARMS_rv primer. All samples display a ycf1b control band with a fragment size of around 950 basepairs. An additional diagnostic ARMS band with a fragment size of 800 basepairs is present in all African individuals (labelled yellow) and also the Arabic M. peregrina (labelled orange). This specific Moringa band (excluding M. oleifera) is absent in M. oleifera and all Moringa commercial products.