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DNA TRACEABILITY FOR VARIETY
PURITY IN NESPRESSO PRODUCT
MOREL E.1, BELLANGER L.1, LEFEBVRE-PAUTIGNY F.2, LAMBOT C.1, CROUZILLAT D.1.
1: Nestlé R&D Center, France. 2: Nestlé Nespresso, Lausanne, Switzerland.
INTRODUCTION The coffee market is regularly developing finished products based on a single variety. Some of them are well recognized by the consumer for example
Maragogype, Moka, Blue Mountain or Bourbon. The development of this product category affords some similarities with the wine market which is organized by
grape varieties in many countries and could bring opportunities for premium product differentiation. Nevertheless, the green coffee market is not organized by
varieties but mainly by species (Robusta versus Arabica) and by producing countries. The distinction of coffee varieties at the green coffee stage is almost
impossible using physical or chemical analytical tools. A DNA method was developed to allow the identification of varieties through the value chain, from the
field to the finished product. The method is applied on routine basis to guarantee the purity and authenticity of raw material used by Nespresso.The quality
control test was recently applied and fine-tuned using green bean batches from farms in Southern Brazil, which grow red and yellow bourbon varieties. This
Arabica blend is at the origin for the sensory specificity of Dulsão do Brasil capsule. Genetic diversity studies were performed among these farms using a set of
eight microsatellite markers (SSRs) selected for their ability to discriminate the Bourbon origins. The DNA tool helped guide the farm selection. The same
procedure could be applied to other Arabica varieties according to requirements and claims of the final coffee product. The method is being improved to increase
the capacity of sample analysis and decrease its cost. In addition, a new technology is under test based on high throughput 454 DNA sequencing. This assay will
provided higher reliability and accuracy both for genetic diversity studies and quality control tests.
MATERIALS & METHODS DNA Extraction of green coffee seeds: 90 green beans per sample are individually and finely cut, pieces of seeds are collected in collection microtubes. The DNA
extraction was performed with the Qiagen kit (DNeasy 96 Plant) following the recommendations of the supplier.
DNA extraction from pool of beans: For each batch, a sample of 1000 coffee beans is grinding then DNA extraction was performed using previous Qiagen protocol.
Selection of SSRs markers: Microsatellites (30 markers) were used to build a genetic database on Arabica strategic varieties already identified by Nespresso (Bourbon Red,
Amarello, Kona, …). They have been selected according to their rate of polymorphism in C. arabica cultivated varieties.
Microsatellite analysis on 3500 xL Genetic Analyzer: Amplified DNA products, by PCR, were separated on the sequencer using capillary electrophoresis according to their
sizes and to the fluorescent labelling. Experimental data were analysed using GeneMapper® software (Applied Biosystems).
High Resolution Melting (HRM): amplification and analysis: HRM is a post PCR method able to detect the genetic variations (SNPs) in PCR amplicons. The amplification
cycles were followed by the high resolution melting steps.
DNA sequencing on Arabica varieties using Roche 454® :, Roche protocol of DNA Amplicon was used to perform this sequencing.
RESULTS
Conclusion
DNA markers are increasingly used by the food industry in the field of traceability of raw materials. This allows offering to consumers a better guarantee on
the Arabica supply chain. New opportunity based on genomic, such as genome sequencing, will be speed up QC for coffee specialties.
NSG : Genomic Analysis
Import Raw Data
(454/ Illumina)
Remove Barcodes and group
(Multiplexing)
Trim sequences
Map Reads to reference
Identification of DIPs
Identification of SNPs
Comparative analysis
Genomics Gateway
Bourbon reference
Adulterant Type 3
Adulterant Type 1
Adulterant Type 2
MID2 Map
Reference
MID6 Map
ADULT. 1
MID7 Map
ADULT. 2
MID7 SNPs
MID6 SNPs
MID2 SNPs
MID7 DIPs
MID6 DIPs
MID2 DIPs
HRM Analysis
SSRs Analysis
CAAGGGTAAAACTGTCCAAGAAGGAAAGTGGGGGAAATGTTTGTCGTGATCATCAATGTCGTGATCGGGAGCTTTTCTGGCAAATTCCTGCCTT Reference
CAAGGGTAAAACTGTCCAAGAAGGAAAGTGGGGGAAATGTTTGTCGCGATCATCAATGTCGTGATCGGGAGCTTTTCTGGCAAATTCCTGCCTT Adulterant 1
CAAGGGTAAAACTGTCCAAGAAGGAAAGTGGGGGAAATGTTTGTCGCGATCATCAATGTCGTGATCGGGAGCTTTTCTGGCAAATTCCTGCCTT Adulterant 2
CAAGGGTAAAACTGTCCAAGAAGGAAAGTGGGGGAAATGTTTGTCGTGATCATCAATGTCGTGATCGGGAGCTTTTCTGGCAAATTCCTGCCTT Adulterant 3
ATTCACTCATTAATTTCCAATGTAACGTTCTAAATCTCACACACACACACACACAC----------------GCAGAGAGCACCGCCAATTCTTCCTATAAAA Reference
ATTCACTCATTAATTTCCAATGTAACGTTCTAAATCACACACACACACACACACACACACACACGCAGAGAGCACCGCCAATTCTTCCTATAAAA Adulterant 1
ATTCACTCATTAATTTCCAATGTAACGTTCTAAATCACACACACACACACACACAC----------------GCAGAGAGCACCGCCAATTCTTCCTATAAAA Adulterant 2
ATTCACTCATTAATTTCCAATGTAACGTTCTAAATCTCACACACACACACACAC--------------------GCAGAGAGCACCGCCAATTCTTCCTATAAAA Adulterant 3
DNA TRACEABILITY
This technique allows to differentiate the specific raw material
composition of green coffee in order to ensure the genetic origin
(Arabica). Genetic analysis with microsatellites give reliable
results for detecting the presence of adulterants. From raw
material, it is possible to determine the reference plant profile for a
specific Arabica variety.
DNA fingerprinting on the main cultivated Arabica varieties allows
the construction of an Arabica Database where the genetic
profiles are recorded.
A routine quality control based on the Arabica genetic diversity
was established in order to trace the raw material from field to
capsules. The adulterant varieties can be detected thanks to the
SSR markers.
HRM (High Resolution Melting) allow to analyse genetic
diversity. This technology give reliable and fast results in
few time. The main application for HRM is the
identification of variants in order to differentiate off types
among Arabica varieties.
This reliability allows to assess a true adulterant rate for
Arabica batches.
New Sequencing Generation
have been introduced in the
QC workflow.
DNA sequencing authorizes
the exhaustive detection of
variants. We are recording
the main commercial
Arabica varieties origins. An
Arabica database will
manage the different DNA
polymorphism (SNP, DIPs…)
found in these varieties.
One perspective related to
the use of this genomic
technology is to create new
tools for traceability such as
DNA chip. This technology
will combined in a single
analysis all the genomic
information for Arabica QC.