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.