Abstract
Saffron (Crocus sativus L.) is very expensive and, because of this, often subject to adulteration. Modern genetic fingerprinting techniques are an alternative low cost technology to the existing chemical techniques, which are used to control the purity of food products. Buddleja officinalis Maxim, Gardenia jasminoides Ellis, Curcuma longa L., Carthamus tinctorius L. and Calendula officinalis L. are among the most frequently-used adulterants in saffron spice. Three commercial kits were compared concerning the ability to recover PCR-grade DNA from saffron, truly adulterated samples and possible adulterants, with a clear difference among them, mainly with the processed samples. Only one of the three kits was able to obtain amplifiable DNA from almost all of the samples, with the exception of extracts. On the recovered DNA, new markers were developed based on the sequence of the plastid genes matK and rbcL. These primers, mainly those developed on matK, were able to recognize saffron and the adulterant species and also in mixtures with very low percentages of adulterant. Finally, considering that the addition of different parts of saffron flowers is one of the most widespread adulterations, by analyzing the DNA of the different parts of the flower (styles, stamens and tepals) at the genetic and epigenetic level, we succeeded in finding differences between the three tissues that can be further evaluated for a possible detection of the kind of fraud.
Highlights
Saffron is an ancient spice that consists of the dehydrated stigmas of the triploid sterile plantCrocus sativus Linn. [1]
In order to continue implementation of the existing methodologies to detect the presence of the main plant adulterants in saffron production, the present work has been focused on the comparison of different DNA extraction methods to better recover DNA from the considered matrices and on the development of DNA markers to identify the presence, in saffron production, of adulterants, such as Buddleja officinalis, Gardenia jasminoides, Curcuma longa, Carthamus tinctorius and Calendula officinalis
Several obstacles must be faced when working with DNA from food and processed samples, in particular: the presence of inhibitors that can hinder enzymatic reactions, such as PCR [11,12], and the DNA that is usually recoverable from these matrices can be very poor and highly degraded [13]
Summary
Crocus sativus Linn. [1]. The spice, other than for culinary uses and colorant properties, contains different secondary metabolites, and because of this, it has been historically used in traditional medicine for a number of health properties, many of which have been scientifically confirmed or Molecules 2016, 21, 343; doi:10.3390/molecules21030343 www.mdpi.com/journal/molecules. Different adulterations have been detected, involving the addition of different plant species, animal-derived substances, synthetic dyes and chalk, among others [4,5] Nowadays, topics such as food authenticity, genuineness and the detection of adulteration in food products, usually economically motivated, are increasingly important for consumers, regulatory agencies and the food industry [6] Presently, within the most-frequently reported plant materials used to adulterate saffron, there are: (1) cut and/or dyed C. sativus stamens; (2) safflower and calendula petals (Carthamus tinctorius L. and Calendula officinalis L.); (3) curcuma powdered rhizomes (Curcuma longa L.); (4) gardenia yellow from. In order to continue implementation of the existing methodologies to detect the presence of the main plant adulterants in saffron production, the present work has been focused on the comparison of different DNA extraction methods to better recover DNA from the considered matrices and on the development of DNA markers to identify the presence, in saffron production, of adulterants, such as Buddleja officinalis, Gardenia jasminoides, Curcuma longa, Carthamus tinctorius and Calendula officinalis. Considering that adulteration can be carried out by adding different parts of the crocus flower itself, the comparison of the MS-AFLP (methyl-sensitive-amplified fragment length polymorphism) and AFLP (amplified fragment length polymorphism) profiles of tepals, stamens and stigmas has been carried out in order to show that polymorphic signals are potentially useful for traceability purposes
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