Tc-MYBPA an Arabidopsis TT2-like transcription factor and functions in the regulation of proanthocyanidin synthesis in Theobroma cacao.

BACKGROUND: The flavan-3-ols catechin and epicatechin, and their polymerized oligomers, the proanthocyanidins (PAs, also called condensed tannins), accumulate to levels of up to 15 % of the total weight of dry seeds of Theobroma cacao L. These compounds have been associated with several health benefits in humans. They also play important roles in pest and disease defense throughout the plant. In Arabidopsis, the R2R3 type MYB transcription factor TT2 regulates the major genes leading to the synthesis of PA. RESULTS: To explore the transcriptional regulation of the PA synthesis pathway in cacao, we isolated and characterized an R2R3 type MYB transcription factor MYBPA from cacao. We examined the spatial and temporal gene expression patterns of the Tc-MYBPA gene and found it to be developmentally expressed in a manner consistent with its involvement in PAs and anthocyanin synthesis. Functional complementation of an Arabidopsis tt2 mutant with Tc-MYBPA suggested that it can functionally substitute the Arabidopsis TT2 gene. Interestingly, in addition to PA accumulation in seeds of the Tc-MYBPA expressing plants, we also observed an obvious increase of anthocyanidin accumulation in hypocotyls. We observed that overexpression of the Tc-MYBPA gene resulted in increased expression of several key genes encoding the major structural enzymes of the PA and anthocyanidin pathway, including DFR (dihydroflavanol reductase), LDOX (leucoanthocyanidin dioxygenase) and BAN (ANR, anthocyanidin reductase). CONCLUSION: We conclude that the Tc-MYBPA gene that encodes an R2R3 type MYB transcription factor is an Arabidopsis TT2 like transcription factor, and may be involved in the regulation of both anthocyanin and PA synthesis in cacao. This research may provide molecular tools for breeding of cacao varieties with improved disease resistance and enhanced flavonoid profiles for nutritional and pharmaceutical applications.

Proanthocyanidin synthesis in Theobroma cacao: genes encoding anthocyanidin synthase, anthocyanidin reductase, and leucoanthocyanidin reductase.

BACKGROUND: The proanthocyanidins (PAs), a subgroup of flavonoids, accumulate to levels of approximately 10% total dry weight of cacao seeds. PAs have been associated with human health benefits and also play important roles in pest and disease defense throughout the plant. RESULTS: To dissect the genetic basis of PA biosynthetic pathway in cacao (Theobroma cacao), we have isolated three genes encoding key PA synthesis enzymes, anthocyanidin synthase (ANS), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). We measured the expression levels of TcANR, TcANS and TcLAR and PA content in cacao leaves, flowers, pod exocarp and seeds. In all tissues examined, all three genes were abundantly expressed and well correlated with PA accumulation levels, suggesting their active roles in PA synthesis. Overexpression of TcANR in an Arabidopsis ban mutant complemented the PA deficient phenotype in seeds and resulted in reduced anthocyanidin levels in hypocotyls. Overexpression of TcANS in tobacco resulted in increased content of both anthocyanidins and PAs in flower petals. Overexpression of TcANS in an Arabidopsis ldox mutant complemented its PA deficient phenotype in seeds. Recombinant TcLAR protein converted leucoanthocyanidin to catechin in vitro. Transgenic tobacco overexpressing TcLAR had decreased amounts of anthocyanidins and increased PAs. Overexpressing TcLAR in Arabidopsis ldox mutant also resulted in elevated synthesis of not only catechin but also epicatechin. CONCLUSION: Our results confirm the in vivo function of cacao ANS and ANR predicted based on sequence homology to previously characterized enzymes from other species. In addition, our results provide a clear functional analysis of a LAR gene in vivo.

Impact of fermentation, drying, roasting and Dutch processing on flavan-3-ol stereochemistry in cacao beans and cocoa ingredients.

This paper reports a systematic study of the level of flavan-3-ol monomers during typical processing steps as cacao beans are dried, fermented and roasted and the results of Dutch-processing. Methods have been used that resolve the stereoisomers of epicatechin and catechin. In beans harvested from unripe and ripe cacao pods, we find only (-)-epicatechin and (+)-catechin with (-)-epicatechin being by far the predominant isomer. When beans are fermented there is a large loss of both (-)-epicatechin and (+)-catechin, but also the formation of (-)-catechin. We hypothesize that the heat of fermentation may, in part, be responsible for the formation of this enantiomer. When beans are progressively roasted at conditions described as low, medium and high roast conditions, there is a progressive loss of (-)-epicatechin and (+)-catechin and an increase in (-)-catechin with the higher roast levels. When natural and Dutch-processed cacao powders are analyzed, there is progressive loss of both (-)-epicatechin and (+)-catechin with lesser losses of (-)-catechin. We thus observe that in even lightly Dutch-processed powder, the level of (-)-catechin exceeds the level of (-)-epicatechin. The results indicate that much of the increase in the level of (-)-catechin observed during various processing steps may be the result of heat-related epimerization from (-)-epicatechin. These results are discussed with reference to the reported preferred order of absorption of (-)-epicatechin > (+)-catechin > (-)-catechin. These results are also discussed with respect to the balance that must be struck between the beneficial impact of fermentation and roasting on chocolate flavor and the healthful benefits of chocolate and cocoa powder that result in part from the flavan-3-ol monomers.

Cacao seeds are a "Super Fruit": A comparative analysis of various fruit powders and products.

BACKGROUND: Numerous popular media sources have developed lists of "Super Foods" and, more recently, "Super Fruits". Such distinctions often are based on the antioxidant capacity and content of naturally occurring compounds such as polyphenols within those whole fruits or juices of the fruit which may be linked to potential health benefits. Cocoa powder and chocolate are made from an extract of the seeds of the fruit of the Theobroma cacao tree. In this study, we compared cocoa powder and cocoa products to powders and juices derived from fruits commonly considered "Super Fruits". RESULTS: Various fruit powders and retail fruit products were obtained and analyzed for antioxidant capacity (ORAC (µM TE/g)), total polyphenol content (TP (mg/g)), and total flavanol content (TF (mg/g)). Among the various powders that were tested, cocoa powder was the most concentrated source of ORAC and TF. Similarly, dark chocolate was a significantly more concentrated source of ORAC and TF than the fruit juices. CONCLUSIONS: Cocoa powder and dark chocolate had equivalent or significantly greater ORAC, TP, and TF values compared to the other fruit powders and juices tested, respectively. Cacao seeds thus provide nutritive value beyond that derived from their macronutrient composition and appear to meet the popular media's definition of a "Super Fruit".

Impact of fermentation, drying, roasting, and Dutch processing on epicatechin and catechin content of cacao beans and cocoa ingredients.

Low molecular weight flavan-3-ols are thought to be responsible, in part, for the cardiovascular benefits associated with cocoa powder and dark chocolate. The levels of epicatechin and catechin were determined in raw and conventionally fermented cacao beans and during conventional processing, which included drying, roasting, and Dutch (alkali) processing. Unripe cacao beans had 29% higher levels of epicatechin and the same level of catechin compared to fully ripe beans. Drying had minimal effect on the epicatechin and catechin levels. Substantial decreases (>80%) in catechin and epicatechin levels were observed in fermented versus unfermented beans. When both Ivory Coast and Papua New Guinea beans were subjected to roasting under controlled conditions, there was a distinct loss of epicatechin when bean temperatures exceeded 70 °C. When cacao beans were roasted to 120 °C, the catechin level in beans increased by 696% in unfermented beans, by 650% in Ivory Coast beans, and by 640% in Papua New Guinea fermented beans compared to the same unroasted beans. These results suggest that roasting in excess of 70 °C generates significant amounts of (-)-catechin, probably due to epimerization of (-)-epicatechin. Compared to natural cocoa powders, Dutch processing caused a loss in both epicatechin (up to 98%) and catechin (up to 80%). The epicatechin/catechin ratio is proposed as a useful and sensitive indicator for the processing history of cacao beans.

Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders.

BACKGROUND: The objective of this study was to validate an improved 4-dimethylaminocinnamaldehyde (DMAC) colorimetric method using a commercially available standard (procyanidin A2), for the standard method for quantification of proanthocyanidins (PACs) in cranberry powders, in order to establish dosage guidelines for the uropathogenic bacterial anti-adhesion effect of cranberry. RESULTS: Commercially available cranberry samples were obtained (five from U.S. sources and six from European sources) for PAC quantification in five different analytical laboratories. Each laboratory extracted and analyzed the samples using the improved DMAC method. Within-laboratory variation (mean +/- SD) was 4.1 +/- 1.7% RSD (range, 2.3-6.1% RSD) and the between laboratory variability was 16.9 +/- 8.5% RSD (range, 8-32% RSD). For comparative purposes, the cranberry samples were alternatively quantified using weights of extracted PACs (gravimetric). The correlation coefficient between the two methods was 0.989. CONCLUSION: This improved DMAC method provides a simple, robust and relatively specific spectrophotometric assay for total PACs in cranberry samples using commercially available procyanidin A2 dimer as a standard. DMAC is most useful within a given type of food such as cranberries, but may not be appropriate for comparing concentrations across different food types, particularly in those cases where large differences exist among the relative amounts of each oligomer and polymer.

Determination of total procyanidins in selected chocolate and confectionery products using DMAC.

A simple, specific, high-throughput colorimetric method based on the reaction of 4-dimethylaminocinnamaldehyde (DMAC) with flavan-3-ols was developed to determine total procyanidins in selected cacao-based products. Extracts of defatted samples were dispensed into a 96-well plate and reacted with DMAC. The absorbance of the reaction products was measured at 640 nm and compared to commercially available procyanidin B2 as a standard. The use of the 96-well plates and a plate reader dramatically improved sample throughput. A standard protocol was established and used for further studies. The calibration was found to be linear from 1-100 ppm. The DMAC reagent reacted relatively specifically to (-)-epicatechin, (+)-catechin, epigallocatechin, gallocatechin, the gallates of catechin, epicatechin, gallocatechin, and epigallocatechin, oligomeric procyanidins of cocoa up to n=4, and A-type procyanidins. Little or no reaction occurred with cyanidins and representative compounds of phenolic acids, flavones, flavanones, flavonols, anthocyanidins, isoflavones, and stilbenes. Sample precision studies were carried out on 10 different test materials over several weeks, and yielded RSD values of 4.0 to 9.5%. The method was ring-tested in three laboratories using blinded test materials including cocoa beans, cocoa powder, chocolate liquor, dark chocolate, and milk chocolate. There was excellent agreement of the results between laboratories.

Characterization of primary standards for use in the HPLC analysis of the procyanidin content of cocoa and chocolate containing products.

This report describes the characterization of a series of commercially available procyanidin standards ranging from dimers DP = 2 to decamers DP = 10 for the determination of procyanidins from cocoa and chocolate. Using a combination of HPLC with fluorescence detection and MALDI-TOF mass spectrometry, the purity of each standard was determined and these data were used to determine relative response factors. These response factors were compared with other response factors obtained from published methods. Data comparing the procyanidin analysis of a commercially available US dark chocolate calculated using each of the calibration methods indicates divergent results and demonstrate that previous methods may significantly underreport the procyanidins in cocoa-containing products. These results have far reaching implications because the previous calibration methods have been used to develop data for a variety of scientific reports, including food databases and clinical studies.

Stability of cocoa antioxidants and flavan-3-ols over time.

Several recent reports have been published indicating that the antioxidant activity of olive oil and tea leaves is not stable over product shelf lives of about one year. We have measured the antioxidant activity, total polyphenols, flavan-3-ols monomers, and procyanidin levels in milk and dark chocolate, in cocoa powder, and in cocoa beans. Results show that for the cocoa products studied, antioxidant activity, and flavan-3-ol levels are stable over typical shelf lives of one year under controlled storage and over 2 years in ambient storage in the laboratory. We also show that 80 year old cocoa powder and 116 year old cocoa beans still show very high levels of antioxidant activity and flavan-3-ol content.

Impact of alkalization on the antioxidant and flavanol content of commercial cocoa powders.

Cocoa is a food ingredient that is important for the contribution of flavor to foods but is also associated with potential health benefits. The chemistry thought to be responsible for its cardiovascular health benefits is the flavanol (flavan-3-ol) antioxidants. Evidence from the literature indicates that natural cocoas are high in flavanols, but when the cocoa is processed with alkali, also known as Dutch processing or Dutching, the flavanols are substantially reduced. This paper provides a survey of the physical and chemical composition of representative natural cocoas and lightly, medium, and heavily alkalized cocoas. As part of the survey, both brown/black and red/brown alkali-processed cocoas were measured. Natural cocoa powders have an extractable pH of 5.3-5.8. Alkalized cocoa powders were grouped into lightly treated (pH 6.50-7.20), medium-treated (pH 7.21-7.60), and heavily treated (pH 7.61 and higher). The natural, nonalkalized powders had the highest ORAC and total polyphenols and flavanols (including procyanidins). These chemical measurements showed a linear decrease as the extractable pH of the cocoa powder increased. Likewise, the flavanol monomers, oligomers, and polymers all showed a linear decrease with increasing pH of the final cocoa powder. When brown/black cocoa powders were compared to red cocoa powders, similar decreases in flavanols were observed with increased alkalization. The average total flavanol contents were 34.6 +/- 6.8 mg/g for the natural cocoas, 13.8 +/- 7.3 mg/g for the lightly processed cocoas, 7.8 +/- 4.0 mg/g for the medium processed cocoas, and 3.9 +/- 1.8 mg/g for the heavily processed cocoa powders. The observed linear and predictable impact of alkalization on flavanol content is discussed with respect to other reports in the literature as well as what implications it may have on diet and food manufacturing.