Food forensics is the science that applies molecular biological methods to analyze food origin and detect frauds. Food adulteration has always been a problem throughout history, particularly for niche or highly valuable products. The determination of new and more sensible methods for the traceability of food and feed, is a continuous arms race between institutions and fraudsters.

Most common frauds are cut or complete substitution of food items with less valuable products, or the falsification of marks of origin/quality.

Spices have been always subjected to adulteration due to their high price. Saffron is considered by many the most precious and expensive spice of all, and its traceability is becoming more and more important, as the demand for a high-quality saffron increase in the market. Saffron itself is made of dried stigmas collected from a cultivated plant, Crocus sativus, the traditional cultivation method has very low yields and it’s, in part, responsible for its price.

Today’s paper (Vikram Khilare et al, 2019, Food Chemistry) deals with the different kinds of analysis that are used to evaluate the quality of saffron samples (total 36), that were collected in different locations in India.


Types of adulterations

The researchers created 3 categories of the most common saffron frauds:

  1. Substitution with similar materials: herbal materials such as beet fibers, pomegranate fibers, red-dyed silk fibers, or Auto adulteration. The latter consists in using other parts of the same plant (Crocus sativus), different from stigmas.
  2. Augmentation of saffron mass: by soaking the fibers in oils or glycerin.
  3. Use of dyes: natural or artificial. Particularly important when the sample is cut with colorless fibers.

The Analysis

In the paper three different kinds of tests are used, the standard ISO method, a classic microscopic observation, and a new DNA-based approach.

The standard for saffron analysis is the ISO 3632-1:2011, where is reported the standardized method for the classification of saffron in five different quality categories. It is based on the detection of two different metabolites by direct measure of absorbance. Safranal is detected at 443nm, it is a volatile compound responsible for the saffron aroma. Crocin is detected at 308nm, and is responsible for color intensity.

Using direct reading of the absorbance from 1% aqueous solutions of dried saffron, each sample is assign to a particular quality category. Note that the analysis itself is not based on the detection of saffron-unique molecules, and that the analysis itself is pretty rapid and cheap.

The other two analyses were a simple observation under 40x magnification, focusing in particular on the fibers structure and color distribution, the other one was a metabarcoding analysis.

Simple comparison of an authentic saffron stigma (left) and an adulterated sample. Note the differences in fiber structure and the uneven color distribution in the picture on the right (dye aggregates "AD").

Barcodes are DNA markers that are selected to identify unknown species (or other taxonomical levels) present in a particular sample. The sequence is selected among highly conserved genes: the barcode needs to be sufficiently variable to allow the detection at the species level, exploiting the different genes variants.

In this case, a portion of the rbcL (RuBisCO enzyme) gene was selected and amplified through PCR. The amplicon was then sequenced through the Sanger sequencing method, and the barcodes were compared on different databases. Both NCBI and BOLD were used to detect the species present in the sample.


Example of the results given by the database analysis. For each sample one or more species have been identified, for each species, a percentage of identity is given. Higher the identity higher is the match of the unknown sample with that particular species, the e-value is an error value that for good assignments needs to be as low as possible. Full picture in the additional material of the paper.


Results

How many of the samples were adulterated?

By using the barcode analysis as a reference, 10 samples showed the presence of tissue coming from different species and were considered adulterated (2 samples did not recover a usable PCR product). The adulterated samples were also identified by using other methods. In particular, the microscopic analysis revealed easily the presence of extraneous tissues with un-even coloration (artificially dyed).

 

What is the quality of the product in the market?

None of the samples belong to high-quality categories I and II, only 20% of the sample reached category III, 36% were classified as category IV, and the remaining samples didn’t meet the standard and were not classified in any category. The ISO methodology is able to detect true saffron samples even without the exploitation of species-specific compound like DNA or proteins, and can also be used against auto adulteration, because diluted samples will result in lower concentration of metabolites, and so will be assign to lower quality categories.

 

Full article available here

Khilare, V., Tiknaik, A., Prakash, B., Ughade, B., Korhale, G., Nalage, D., ... & Khedkar, G. (2019). Multiple tests on saffron find new adulterant materials and reveal that Ist grade saffron is rare in the market. Food chemistry, 272, 635-642.

Other references:

ISO 3632-1:2011: https://www.iso.org/obp/ui/#iso:std:iso:3632:-1:ed-2:v1:en note that the technical part of ISO standards are not for free.