Cover Photo Credit: Rainis Venta

This is a summary of the paper listed above by Ludlow et al. (2016) which analyzes the genetic strains of various S. cerevisiae related to wine, coffee, and cacao. All tables and figures belong to the publishers of the research paper.

Yeasts, Wine, Coffee, & Cacao!

There’s much information in regards to yeasts associated with wine making. Yeast associated with wine making have been traveling the world for over 9000 years since humans have been making wine. With origins in the Middle East, humans took the methods, the vines, and unknowingly, the yeasts with them everywhere they went. Because of this, the yeast strains of S. cerevisiae associated with wine are genetically very similar worldwide. In the same way, human migration starting from the Northern Amazon has globally dispersed various strains of S. cerevisiae yeasts involved with fermenting cacao beans. This study looks at the various strains of S. cerevisiae yeasts in various countries worldwide, and analyzes how genetically similar or dissimilar they are from one another.

Theobroma cacao originated in the Amazon and Orinoco basins of Colombia and Venezuela. It was then brought to central America somewhere between 3000 and 1800 BC. In 1530, Spaniard Hernan Cortes introduced cacao and chocolate to the Europeans. In the 16th-20th centuries, cacao trees were dispersed into other parts of Central and South America, Asia, Oceania, Africa, and even Australia.

Coffee originated in Ethiopia, and was dispersed throughout the Middle East and North Africa by the Arab traders as early as the 6th Century. It wasn’t introduced into Europe until the 17th Century.

The researchers here found patterns between the historical migration of these plants with the relatedness of the various strains of the yeasts associated with cacao and coffee.

They wanted to test whether the S. cerevisiae yeasts of cacao and coffee fermentation are:

genetically similar or genetically diverse
crop-specific (are coffee yeasts unique only to coffee?)
geography-specific (are yeast strains in Ghana unique to Ghana, or closely related to yeasts in other countries?)

Although this study looks into yeast strains associated with coffee and cacao, I’ll focus mainly on cacao for the purpose of this summary.

isolating & Culturing S. cerevisiae yeasts from cacao

Saccharomyces cerevisiae yeasts were cultured from dried unroasted cacao beans. All the country of origins, listed in Table 1 below, were extracted from 8-10 unroasted cacao beans by the researchers themselves (except from Ghana). The information on the Ghana yeast strains were obtained from already published literature. The cacao beans were obtained from Theo Chocolate (Seattle, WA, USA) and Chocolate Alchemy (Eugene, OR, USA). The cacao beans originated from:

Colombia, Costa Rica, Dominican Republic, Ecuador, Ghana, Haiti, Ivory Coast, Madagascar, Nicaragua, Nigeria, Papua New Guinea, Peru, and Venezuela

Interesting findings from yeast cultures obtained

The researchers state that by analyzing the DNA sequence of yeasts cultured from unroasted beans of an unknown origin, one can more or less accurately pinpoint the geographic origin of where the cacao beans came from! In other words, by knowing the strains of yeast, one can know where the cacao originated!

They also noted that the cocoa bean cultures contained various bacteria, other yeasts, and filamentous fungi as well. They suggest this approach can be adapted to isolate other microorganisms from unroasted cacao and coffee beans as well.

Coffee and Cacao Yeasts form Distinct yet Genetically Diverse Populations

To measure the genetic diversity of the yeasts, the researchers used RAD sequencing (Restriction site associated DNA sequencing) and compared the cacao and coffee strains with wine strains from previously published data.

The results? They note a “sharp contrast” between the yeast strains of wine, coffee, and cacao. You can see for yourself in Figure 1 A below. Each dot is a different yeast sample. Blue represents wine strains, red is coffee, and green is cacao strains. You can see that for the most part, the colours generally don’t overlap too much, with the majority of the dots clustering towards one area (the blue are all clustered in one spot). This indicates how diverse the strains are in regards to type of plant (cacao, coffee, wine), but also how similar they are within their own group.

The yeast strains coming from wine from different regions were genetically much more similar to one another. On the contrary, the coffee and cacao strains were much more genetically diverse (not clustered in one spot, but spread out over a greater area), unlike with wine. See Figure 1 B below to show the degree of genetic diversity. You can see that within coffee and cacao, there is much more “genetic distance” than there is with the wine column.

Crop-specificity

They state that coffee and cacao strains seem to be less “crop-specific” due to their greater genetic diversity, especially relative to wine yeasts. However, they point out that this doesn’t prove there isn’t some crop-specific origins of coffee and cacao yeasts, and more work needs to be done to confirm this.

Origin-specificity

Strains of yeast cultured from cacao beans of various origins.

Cacao and coffee strains showed strong country-level clustering even though the bean samples were obtained from many suppliers from different places and times. See Figure. 1 C (coffee) & D (cacao) above. Each circle, square, or rectangle is a yeast sample. Each colour corresponds with a sample from a specific country. For example, notice how all the yellow dots in Figure 1D (which represent Venezuela cacao samples) are clustered within the same area, and the same for many other samples with some exceptions. You can see that some coloured dots cluster more closely (more genetically similar) and some coloured dots are spread out a bit more, even overlapping other clusters. These yeasts have a bit more genetic diversity, yet still quite similar enough to point out the country of origin to a degree.

Coffee and cacao yeast strains seem to form their own discreet populations. We can also see from the graph that not all of these yeasts from various origins are related. This suggests independent origins for distinct populations of yeasts, and may be associated with the migration patterns and involvement of humans that may have likely transported various yeasts strains.

These results also support the hypothesis that the yeasts isolated on the cacao beans originated from local fermentation rather than from cross-contamination during shipping and distribution.

Admixture and Migration Events

What’s an Admixture?

An admixture in regards to genetics (genetic admixture), is the presence of DNA in an individual from a distantly-related population or species. This is a result of interbreeding between populations or species who have been reproductively isolated and genetically differentiated.

Cacao & Coffee Admixtures

In this study, the researchers discovered new yeast populations, and wanted to figure out the origins of these new strains. In particular, the strains labeled as “Pan/Mixed 2” and “South American” (SA).

The new coffee and cacao populations were not composed of strains with novel alleles, but instead were made up of an admixture of the three known yeast populations. In other words, these new strains of yeasts found on cacao were a genetic mixture of various other known strains that were otherwise found in other regions/countries. These admixtures roughly corresponded to the geographic proximity of the samples’ origin, and also seem to correspond to the patterns of human migration.

Figure 2 below shows a plot of various yeast populations, some found in this study, but most from previously published data. Each strain of yeast is colour coded, and plotted out with Multidimensional scaling (MDS). MDS is a way to visualize the genetic distance or similarity each sample has. Notice the wide distance range of the Europe/Vineyard strain, confirming that yeasts associated with wine making are much more genetically similar regardless of where in the world it is.

The researchers state that their results provide evidence that suggests these admixtures correspond to the geographic proximity of the samples’ origin as well as with patterns of human migration. The two South American populations of yeasts (one coffee one cacao) share alleles with North American oak population (the name of a strain of yeast found in North America). Cacao originated in South America, transported up to Mexico and Southwestern United States before being widely dispersed by Europeans. The African yeast populations show mixtures with European and Asian alleles, suggesting migration of yeasts between Africa and Europe and Africa and Asia (Figure 3).

They also state the history of global traffic in “yeasts associated with wine fermentation may have set the stage for subsequent mingling and admixture events that gave rise to these new populations.”

Discussion

Although wine, coffee, and chocolate all rely on humans cultivating, harvesting, and fermenting them, wine differs in a few respects.

The oak barrels used in wine fermentation are often sent from established wine making regions to new areas, and indirectly transport yeasts from one region to another. Cacao beans are fermented in wooden boxes or baskets that are made locally where the cacao is grown.
Wine making often requires starter cultures (clonal populations) to get the fermentation going. Although some growers do inoculate their cacao, it’s been standard practice for generations to for cacao to be spontaneously fermented (from local yeasts, similar to how real sour dough is made).

So, the spontaneous fermentation styles of the cacao suggest that the yeast strains associated with them are not as similar as yeasts found in the region where the plants originated, even though there appears to be some overlap (admixtures). This is what is suggested to be the reason for the greater diversity in cacao yeast genetics. The authors suggest the coffee and cacao yeasts are the result of admixture events, combining alleles from Europe, Asia, and North America.

The results show that the yeasts of cacao fermentation were carried out by populations of yeasts specific to a geographical region. These populations appear to have risen independently.

Yeasts associated with coffee and cacao appear to be the result of admixture events, combining strains of yeasts from Europe, Asia, and North America. Humans may have helped foster the establishment of these hybrid yeast groups.

In several cases, the combinations of alleles present in these groups coincide with known paths of transportation, organized cultivation, and fermentation of the crops.

Future applications of these findings?

The genetic variation found in these new populations of yeasts in cacao and coffee provide a source of phenotypic diversity. These various yeast strains could then be exploited in a way to alter or enhance the quality or flavour of the fermenting cacao and coffee beans.

We know that different wine strains of yeast can produce very different fermentation results. Bokulich et al. demonstrated that microbes found on grapes differ according to cultivar, region, and climate. He described the term “microbial terroir.”

Knight et al. showed that grape fermentations using S. cerevisiae strains isolated from different locations have chemical profiles that correlate with the region of origin. We also know this to be true from sourdough starters, that have a unique taste depending on where the starter was made and the local yeasts that facilitated the process.

When we discuss cacao or chocolate flavour and terroir, many people assume soil or climate, when little to no evidence for this exists. Terroir can also be defined as the impact other organisms have on the plant, which includes microorgansims such as yeasts. The more we understand the microorganisms involved in cacao fermentation, the better equipped we will be at using them to our advantage, improving cacao quality and maybe even improving flavour in a way that can be very cost effective and profitable for cacao growers.

Independent Origins of Yeast Associated with Cacao Fermentation