Marker development for the traceability of certified sustainably produced cacao (Theobroma cacao) in the chocolate industry

Lafargue, Pedro

Abstract

Theobroma cacao (cocoa) is one of the most studied commodities around the world and the source of one of the world’s most consumed and familiar products, chocolate. The multibillion-pound industry has changed to a higher demand for sustainably certified cacao (Rainforest Alliance, UTZ, and Fair Trade) and closer attention is being paid to how this such cacao can be traced. The present work describes a new concept, “From Shelf to Farm & Cooperative”, a study to identify the geographical origin of the fermented cacao beans used to manufacture premium and bulk chocolate products. The research sought to assess how DNA based approaches for traceability of food products can be utilised within the supply chain of cacao and chocolate. To identify the factors that influence cacao traceability and the importance of assessing it in different supply chain systems, multi-disciplinary stakeholders from policy makers, small-scale farmers in South and Central America, to the biggest cacao and chocolate manufacturers in Europe were interviewed. Two stages in chocolate production were identified as key to be screened for tracking implementation: The farm (Stage 1) to identify cacao trees genotype composition and the cooperative (Stage 2) where fermentation of cacao beans occur. A reliable modified cacao DNA extraction protocol was developed using the DNeasy mericon Food Kit which enable higher DNA yield from a range of chocolate products including, for the first time, ‘cocoa butter’. DNA markers characterising the chloroplast genome of T. cacao were assessed to trace back the chocolate to Stage 1 (farm). Reference genotypes from the International Cocoa Quarantine Centre at the University of Reading were screened with 25 chloroplast single sequence repeat (cpSSR) markers revealing a level of DNA polymorphism sufficient to reliably identify lineages below the species level to characterise farms. Allelic proportions for nine cpSSR were quantified and compared in DNA extracted from 116 chocolate samples revealing distinct clustering in single-origin chocolate produced from beans harvested in Peru, Ecuador, Venezuela, Trinidad and Madagascar. In contrast, no differentiation was observed for bulk chocolate samples (Mars, Nestle) and beans originating from Côte d'Ivoire farms thus reflecting the lack of allelic diversity found in cultivars in West Africa. To identify unique biomarkers for Stage 2 (cooperative), the fermentation microbiome was assessed by performing amplicon Illumina sequencing on 47 single origin chocolate using the universal 16S v3-v4 ribosomal region and three housekeeping genes from Acetobacter pasteurianus. Variation in microbiome diversity was characterised with unique Amplicon Sequence Variants (ASV) identified per continent, country and fermentation location for which signature bacterial profile was found to be conserved across years. Markers identified in Stage 1 and Stage 2 can be used for tracking cocoa beans origin. To make these biomarkers applicable in industrial scenarios, it will be essential to create a machine learning model that could recognize the specific markers from multiple regions.

• Thesis Type: Thesis
• Deposit Date: Sep 6, 2020
• Publicly Available Date: Apr 21, 2021
• Keywords: Biomarkers , Cocoa, Chocolate, Sustainability, Traceability, Block-chain, Food Safety, Transparency
• Public URL: https://uwe-repository.worktribe.com/output/6642792
• Award Date: Apr 21, 2021