Roasting effects on phenolic content and free-radical scavenging activities of pulp preconditioned and fermented cocoa (Theobroma cacao) beans

Polyphenols are phytochemicals responsible for the astringency, bitterness, green flavours and antioxidant activities in Theobroma cacao beans. Polyphenols degradation in cocoa beans during roasting is crucial to the flavour outcome and it is influenced by factors such as temperature, time and pod storage. Antioxidants are compounds that help to inhibit oxidation reactions caused by free radicals such as singlet oxygen, superoxide, peroxyl radicals, hydroxyl radicals and peroxynitrite thereby preventing damage to the cells and tissues. Their mechanisms of action include scavenging reactive oxygen and decreasing localised oxygen concentration thereby reducing molecular oxygen’s oxidation potential, metabolising lipid peroxides to non-radical products and chelating metal ions to prevent generation of free radicals in humans. The study aimed at investigating changes in total polyphenols, anthocyanins, o-diphenols and antioxidant activity (free-radical scavenging activities) after roasting of pulp preconditioned and fermented cocoa beans using standard analytical methods. A 4×4 full factorial design with the principal experimental factors as pod storage time (0, 3, 7 and 10 days) and roasting duration (0, 15, 30 and 45 minutes) at 120oC were used to study the changes in the total polyphenols, anthocyanins, o-diphenols and % free-radical scavenging activities of the cocoa beans. Variable decrease in total polyphenols, odiphenols and anthocyanins were observed with increase in pre-conditioning (pod storage time) and roasting duration. However, variable trends were observed for the % free-radical scavenging activities. The total polyphenols, anthocyanins and o-diphenols in the cocoa beans after 45 minutes roasting decreased in the range 132.24 to 57.17 mg/g, 6.71 to 1.07 mg/kg and 15.94 to 8.25 mg/g respectively at all pod storage treatments. The total polyphenols of the fermented, dried and unstored (freshly harvested) cocoa beans was 132.25 mg/g which reduced to 122.14 mg/g (7.6% degradation), 116.721 mg/g (11.7% degradation) and 92.22 mg/g (30.3% degradation) after storage for 3, 7 and 10 days, respectively. The optimum decrease in the % freeradical scavenging activity was 7 days and above of pods storage. Increasing roasting time caused a continuous decrease in the % free-radical scavenging activity from 89.10% to 74.31% after 45 minutes for beans from the unstored (freshly harvested) pods. However, pod storage caused an increase in the % free radical scavenging activities during roasting. Pulp pre-conditioning (pod storage) and roasting duration could be used to reduce the astringency and bitterness caused by polyphenols, o-diphenols and anthocyanins in cocoa beans as well as increase the antioxidant activity imparted by cocoa.Key words: Cocoa, pod storage, roasting, polyphenols

Applying SNP marker technology in the cacao breeding programme in Ghana

In this investigation 45 parental cacao plants and five progeny derived from the parental stock studied were genotyped using six SNP markers to determine off-types or mislabeled clones and to authenticate crosses made in the Cocoa Research Institute of Ghana (CRIG) breeding programme. Investigation was based on the 5\u2019 nuclease SNPassay using Illustra Hot Start mix Ready-To-Go PCR strips and BioTek FLx800TBP Fluorescence Microplate Reader. In a group of six cacao plants labeled as PA150 clones and another five labeled as Pound7, one clone in each group was unambiguously determined as off-type or mislabeled. Similarly, in a cohort of 23 PA7 "clones", four genotypes were differentiated. Cross-checking the fidelity of five progeny from the parental stock under study, it was established that no errors were made in the crossing. The most significant outcome of this study, however, was that out of the four categories of 23 PA7 candidate parental trees only one category can be comparable to the reference clone in the International Cacao Germplasm collection, Trinidad (ICG,T); thus informing the need for further work to find the correct clone among these for the breeding programme. It was thus concluded that thissimple yet cutting-edge genotyping procedure can be used in applied cocoa breeding programmes in a cocoa producing country. This work represents a first step in the genotypic characterisation of the CRIG germplasm collection and Seed Gardens.Au cours de cette recherche, 45 plants de cacao parentaux et 5 descendants d\ue9rivant du stock parental ont \ue9t\ue9 g\ue9notyp\ue9 en utilisant 6 marqueurs SNP, afin de d\ue9terminer les clones mal \ue9tiquet\ue9s et d\u2019authentifier les croisements effectu\ue9s dans le programme d\u2019am\ue9lioration de l\u2019Institut de Recherche sur le Cacao au Ghana (CRIG). Cette \ue9tude a \ue9t\ue9 bas\ue9e sur les 5' nucl\ue9ases SNP en utilisant des bandes PCR "Hot Start mix Ready-To-Go PCR strips" et un Lecteur Microplat \ue0 Fluorescence "BioTek FLx800TBP". Au sein d\u2019un groupe de six plants de cacao \ue9tiquet\ue9 PA150 et d\u2019un autre groupe de cinq \ue9tiquet\ue9 Pound 7, il a \ue9t\ue9 d\ue9termin\ue9 sans ambigu\ueft\ue9 qu\u2019un clone par groupe \ue9tait mal \ue9tiquet\ue9. De fa\ue7on similaire, quatre g\ue9notypes diff\ue9rents ont \ue9t\ue9 identifi\ue9s dans une m\ueame cohorte de clones 23PA7. En v\ue9rifiant la fid\ue9lit\ue9 de cinq descendants issus du stock parental \ue9tudi\ue9, il a \ue9t\ue9 \ue9tabli qu\u2019aucune erreur n\u2019avait \ue9t\ue9 faite lors du croisement. Le r\ue9sultat le plus significatif de cette \ue9tude a \ue9t\ue9 que, sur quatre cat\ue9gories de 23 candidats PA7 de souches parentales, une seule pouvait \ueatre comparable au clone de r\ue9f\ue9rence dans la collection Internationale du Germoplasme de Cacao, Trinidad (ICG,T), d\ue9montrant ainsi la n\ue9cessit\ue9 de travaux suppl\ue9mentaires pour d\ue9terminer le clone exact parmi ceux \ue9voqu\ue9s pr\ue9c\ue9demment. Il a ainsi \ue9t\ue9 conclu que cette m\ue9thode avant-gardiste de g\ue9notypage, pourtantsimple, peut \ueatre utilis\ue9e dans les programmes appliqu\ue9s d\u2019am\ue9lioration du cacao dans un pays producteur. Ce travail repr\ue9sente une premi\ue8re \ue9tape dans la caract\ue9risation g\ue9n\ue9tique de la collection du germoplasme CRIG et jardins semenciers

Verification of genetic identity of introduced cacao germplasm in Ghana using single nucleotide polymorphism (SNP) markers

Accurate identification of individual genotypes is important for cacao (Theobroma cacao L.) breeding, germplasm conservation and seed propagation. The development of single nucleotide polymorphism (SNP) markers in cacao offers an effective way to use a high-throughput genotyping system for cacao genotype verification. In the present study, high-throughput genotyping with SNP markers was used to fingerprint 160 cacao trees in the germplasm collection at the Cocoa Research Institute of Ghana (CRIG). These accessions had been originally introduced from international germplasm collections. The multilocus SNP profiles, generated by the Sequenom Mass Spectrometry platform, were compared with the SNP profiles of reference trees maintained in the international cacao collections. The comparison unambiguously identified mislabeled trees. For materials introduced as hybrid seeds without an available reference genotype, parentage analysis and model-based assignment were applied to verify their recorded parentage and genetic background. Our study shows that a small set of polymorphic SNP markers can provide a robust and accurate result for cacao genotype identification. This protocol can be applied for large-scale genotyping of cacao as well as for many other crops.Keywords: Cacao, conservation, chocolate, DNA fingerprint, molecular marker, tropical plant, off-type, true-to-type, West Africa.African Journal of Biotechnology, Vol 13(21), 2127-213

Optimization of Cocoa Pulp Juice Fermentation with Yeast Starter Cultures of Cocoa Heap Fermentations

ABSTRACT The Cocoa Research Institute of Ghana (CRIG) produces crude ethanol from cocoa sweatings by spontaneous fermentation and distillation. The crude ethanol is further refined and blended into gin and brandy for the local market. The operation is sub-optimal and results in waste of raw materials, energy and time. Following a research carried out on the biodiversity and population of microorganisms involved in heap fermentation, pure cultures of the yeasts became available thus prompting the need to further optimize the process of alcohol production from cocoa pulp juice using starter cultures prepared from microorganisms isolated from cocoa heap fermentations. In these experiments, 10 yeast strains and a combination of some of these strains were used as starter cultures for the fermentation of cocoa pulp juice to produce ethanol. Results from metabolite analyses of this study revealed that Saccharomyces cerevisiae and Issatchenkia orientalis gave maximum ethanol production of 1571.7 mM and 1396.8 mM within 36 and 48 h of fermentation, respectively as compared to the spontaneous fermentation which gave maximum ethanol production of 503.2 mM within 48 h. Pichia kluyveri which gave the lowest ethanol production (324.9 mM) produced the maximum mannitol concentration of 249.0 mM

The use of gamma radiation for the preservation of kola nuts

Kola nuts wrapped with Mitragyna stipulosa (Akan: subaha) leaves and stored in baskets (traditional method) and irradiated with Co-60 gamma rays with absorbed doses ranging from 0.10 to 0.50 kGy at a dose rate of 0.171 kGy/hr to control kola weevils (Balanogastris kolae) during storage. The number of nuts per treatment ranged between 20 and 1353, and the ambient storage temperature fluctuated between 16.5EC and 37.5E C. Gamma radiation suppressed the development and emergence of kola weevils that destroy the nuts in storage. Whilst no live insects were found in the treated samples, up to 48 live weevils were counted per packet in the control treatment at 28 days in storage. Consequently, nut infestation in the control was about 98.4%. Fungal deterioration of both irradiated and unirradiated kola nuts was observed. Weight loss in kola nuts irradiated at 0.25 and 0.50 kGy and stored for 84 days after irradiation was 18.2% and 19.2%, respectively, whilst comparable loss from the control treatment was 39.9%. Weevils fed with treated and untreated nuts did not show any preferences and differences in growth. A panel of tasters did not detect any differences between treated and untreated nuts. JOURNAL OF THE GHANA SCIENCE ASSOCIATION Volume 2 No. 3 (2000) pp. 184-19

The impact of SNP fingerprinting and parentage analysis on the effectiveness of variety recommendations in cacao

Published online: 26 April 2015Evidence for the impact of mislabeling and/or pollen contamination on consistency of field performance has been lacking to reinforce the need for strict adherence to quality control protocols in cacao seed garden and germplasm plot management. The present study used SNP fingerprinting at 64 loci to examine the diversity, labeling errors and parentage in 2551 trees obtained from six seed gardens, breeders clone collection and single-cross progenies and a sample of farmers’ trees in Ghana. Clone mislabeling was pervasive, both within the seed garden clones and among clones of the breeders’ active collection. Among the seed garden clones, mislabeled trees were assigned to other parental clones used in the seed garden, pointing to labeling errors prior to planting as the principal cause of mislabeling. Among the breeders’ clone collection, both homonymous and synonymous mislabeling were identified in addition to trees with unique genotypes. This implicates pre-planting labeling errors and rootstocks overtaking budded scions. Parentage analysis supported the Amelonado ancestry of farmers’ varieties but with significant contribution of Upper Amazon introductions. Parentage of recently developed clones and of progenies of controlled crosses showed evidence of both pollen contamination and effects of mislabeled parents. The observed patterns of unexpected parentage had direct effects on the consistency of the variety performance between trials and increased within-plot variability for families with mixed ancestry. The results provide a strong basis for mainstreaming SNP fingerprinting in cacao breeding programs to improve the efficiency of the variety development process.Evidence for the impact of mislabeling and/or pollen contamination on consistency of field performance has been lacking to reinforce the need for strict adherence to quality control protocols in cacao seed garden and germplasm plot management. The present study used SNP fingerprinting at 64 loci to examine the diversity, labeling errors and parentage in 2551 trees obtained from six seed gardens, breeders clone collection and single-cross progenies and a sample of farmers’ trees in Ghana. Clone mislabeling was pervasive, both within the seed garden clones and among clones of the breeders’ active collection. Among the seed garden clones, mislabeled trees were assigned to other parental clones used in the seed garden, pointing to labeling errors prior to planting as the principal cause of mislabeling. Among the breeders’ clone collection, both homonymous and synonymous mislabeling were identified in addition to trees with unique genotypes. This implicates pre-planting labeling errors and rootstocks overtaking budded scions. Parentage analysis supported the Amelonado ancestry of farmers’ varieties but with significant contribution of Upper Amazon introductions. Parentage of recently developed clones and of progenies of controlled crosses showed evidence of both pollen contamination and effects of mislabeled parents. The observed patterns of unexpected parentage had direct effects on the consistency of the variety performance between trials and increased within-plot variability for families with mixed ancestry. The results provide a strong basis for mainstreaming SNP fingerprinting in cacao breeding programs to improve the efficiency of the variety development process.Evidence for the impact of mislabeling and/or pollen contamination on consistency of field performance has been lacking to reinforce the need for strict adherence to quality control protocols in cacao seed garden and germplasm plot management. The present study used SNP fingerprinting at 64 loci to examine the diversity, labeling errors and parentage in 2551 trees obtained from six seed gardens, breeders clone collection and single-cross progenies and a sample of farmers’ trees in Ghana. Clone mislabeling was pervasive, both within the seed garden clones and among clones of the breeders’ active collection. Among the seed garden clones, mislabeled trees were assigned to other parental clones used in the seed garden, pointing to labeling errors prior to planting as the principal cause of mislabeling. Among the breeders’ clone collection, both homonymous and synonymous mislabeling were identified in addition to trees with unique genotypes. This implicates pre-planting labeling errors and rootstocks overtaking budded scions. Parentage analysis supported the Amelonado ancestry of farmers’ varieties but with significant contribution of Upper Amazon introductions. Parentage of recently developed clones and of progenies of controlled crosses showed evidence of both pollen contamination and effects of mislabeled parents. The observed patterns of unexpected parentage had direct effects on the consistency of the variety performance between trials and increased within-plot variability for families with mixed ancestry. The results provide a strong basis for mainstreaming SNP fingerprinting in cacao breeding programs to improve the efficiency of the variety development process

Applying SNP marker technology in the cacao breeding programme in Ghana

In this investigation 45 parental cacao plants and five progeny derived from the parental stock studied were genotyped using six SNP markers to determine off-types or mislabeled clones and to authenticate crosses made in the Cocoa Research Institute of Ghana (CRIG) breeding programme. Investigation was based on the 5’ nuclease SNPassay using Illustra Hot Start mix Ready-To-Go PCR strips and BioTek FLx800TBP Fluorescence Microplate Reader. In a group of six cacao plants labeled as PA150 clones and another five labeled as Pound7, one clone in each group was unambiguously determined as off-type or mislabeled. Similarly, in a cohort of 23 PA7 "clones", four genotypes were differentiated. Cross-checking the fidelity of five progeny from the parental stock under study, it was established that no errors were made in the crossing. The most significant outcome of this study, however, was that out of the four categories of 23 PA7 candidate parental trees only one category can be comparable to the reference clone in the International Cacao Germplasm collection, Trinidad (ICG,T); thus informing the need for further work to find the correct clone among these for the breeding programme. It was thus concluded that thissimple yet cutting-edge genotyping procedure can be used in applied cocoa breeding programmes in a cocoa producing country. This work represents a first step in the genotypic characterisation of the CRIG germplasm collection and Seed Gardens.Au cours de cette recherche, 45 plants de cacao parentaux et 5 descendants dérivant du stock parental ont été génotypé en utilisant 6 marqueurs SNP, afin de déterminer les clones mal étiquetés et d’authentifier les croisements effectués dans le programme d’amélioration de l’Institut de Recherche sur le Cacao au Ghana (CRIG). Cette étude a été basée sur les 5' nucléases SNP en utilisant des bandes PCR "Hot Start mix Ready-To-Go PCR strips" et un Lecteur Microplat à Fluorescence "BioTek FLx800TBP". Au sein d’un groupe de six plants de cacao étiqueté PA150 et d’un autre groupe de cinq étiqueté Pound 7, il a été déterminé sans ambiguïté qu’un clone par groupe était mal étiqueté. De façon similaire, quatre génotypes différents ont été identifiés dans une même cohorte de clones 23PA7. En vérifiant la fidélité de cinq descendants issus du stock parental étudié, il a été établi qu’aucune erreur n’avait été faite lors du croisement. Le résultat le plus significatif de cette étude a été que, sur quatre catégories de 23 candidats PA7 de souches parentales, une seule pouvait être comparable au clone de référence dans la collection Internationale du Germoplasme de Cacao, Trinidad (ICG,T), démontrant ainsi la nécessité de travaux supplémentaires pour déterminer le clone exact parmi ceux évoqués précédemment. Il a ainsi été conclu que cette méthode avant-gardiste de génotypage, pourtantsimple, peut être utilisée dans les programmes appliqués d’amélioration du cacao dans un pays producteur. Ce travail représente une première étape dans la caractérisation génétique de la collection du germoplasme CRIG et jardins semenciers