October 31, 2020

Having gone through the initial trials for cacao protein extractions, I’ve come to a point where I need to organize my ideas on the page.

As I related in an earlier post, the objective of these extractions is to isolate the cacao storage protein, the globular vicilin, and the two enzymes that digest it from the raw unfermented cacao seed. Then, I’ll proceed with several in-vitro reactions to model how the vicilin is digested at specific reaction sites and responds to different pH and organic acid mixtures.

Although I have been following extraction protocols laid out by (Janek, Niewienda, Wöstemeyer, & Voigt (2016); Voigt, Voigt, Heinrichs, Wrann, & Biehl (1994); and Voigt, Textoris-Taube, & Wöstemeyer (2018), there were still quite a few kinks to work out through practice. I practiced these protocols several times. All my practice occurred on fermented unroasted beans; these materials were very accessible and much cheaper than beans cut from raw pods. We wanted to refine the procedure before working with the more expensive raw materials.

For one thing, I had to learn how to use a Soxhlet extraction device and a Rotovap. Both of these became intuitive with time but required a learning curve for someone with no organic chemistry experience.

Secondly, it took me quite a bit of trial and error to figure out the right way to prepare the beans. Early extraction attempts with pentane only returned a few percent cacao fats, indicating that the extraction efficiency was very low. I tested a number of solvents, including pentane, hexane, chloroform, diethyl ether, and pure acetone. Still, pentene always gave the highest return, so eventually, I eliminated the solvent type as the issue and realized that the solvent simply didn’t have enough surface area. I began looking for a better way to crush the beans.

Eventually, we hit on a method of grinding and sifting through a fine sieve. The practice beans were dry enough (7% moisture) that grinding them directly worked well; flash-freezing in liquid nitrogen worked even better. We chose to follow a protocol that suggests flash-freezing and then lyophilizing the materials before grinding and sifting for the* real* materials.

Materials prepared in this way run through several cycles in the Soxhlet. Our reference protocols indicate that cycles should run for 24 hours, but we have conducted tests that find returns diminish to nearly undetectable amounts after about six cycles with the beans prepared in this manner. So, after 6 to 8 cycles of pentane to pull out the fats, we move on to a similar number of chloroform cycles to remove the alkaloids, followed by pure acetone to precipitate the polyphenols.

At this point, we dry the remaining solids in a low oven, producing what is known as Acetone Dry Powder—the material from which I will isolate the proteins and enzymes.

The isolation process, which I have started and have some thoughts about, will have to wait for a different entry. Right now, I’d like to muse about some concerns in my materials preparation. Two main things still concern me.

Firstly, I’m not convinced we should move directly to 100% acetone after the chloroform cycles. The older protocols start with a 70% acetone solution, move up to an 80% solution, and only after several extractions with each of these do they use a 100% acetone solution. My organic chemistry mentor assures me that he is unaware of a suitable chemical reason to step up the acetone concentration, so he thinks we can safely skip doing so. However, I’m still pretty worried about this because of the protein-binding capability of polyphenols. The purpose of acetone steps is to clear out all polyphenols, and it is crucial to eliminate as many as possible. Any polyphenols remaining in the acetone dry powder (ADP) will precipitate precious protein volume, removing it from my extract. Also, polyphenols are water-soluble, so it makes sense that having a mixture of organic solvent and a gentle saline solution would pull out more polyphenols than acetone alone. I think this is part of the protocol that may need to change. We may need to try a small amount of Acetone Dry Powder with the step-wise acetone solutions and a small amount without to test the degree of polyphenol extraction.

This brings me to my second concern with this process. I don’t think I have the efficiency nailed down very well yet. During trial runs, we ran the Rotovap on collection vessels, then weighed the vessels with the contents of the collected extract at each stage (so the fats after pentane, the alkaloids after chloroform, the polyphenols after acetone). After thoroughly cleaning the collection vessel, we weighed it again to get a weight for the collected extract. This seemed like a good idea, except that when I added up all the extract weights, their total value was more than the weight that I had lost from my raw materials. This happened twice. The discrepancy was not enormous numerically, but it is quite significant when you consider the small quantities I am working with. The most likely explanation is that I am not sufficiently drying my collection vessels. I’ll need to seek out some feedback on this.

On one hand, for me, the contents of the collection vessels are waste—I don’t need their contents since my only goal is protein extraction. So, I could quit worrying about this now. But on the other hand, I don’t like the discrepancy in mass because it indicates messiness in my process. I want to clean that up so I can trust myself to do this right. If that means practicing a few more times—well that’s tedious, but that’s what it means.

On the other hand, when it came time to prepare my *real* raw materials from fresh cacao pods, that was a lot of fun. Nine colorful pods arrived, and I hammered them open in the lab. Everyone wanted to taste the fruit, and they all had lots of questions about cacao and chocolate. I used a small knife to break the testa and peel it off, along with the pulp (I wanted to discard the pulp because of its high water content, which would have slowed freeze-drying also because its cellulose was superfluous mass). I dropped the peeled beans into a vat of liquid nitrogen to flash-freeze them immediately. Then, I poured out the whole mass between two sheets of aluminum, where I crushed them with a mallet.

This mass of crushed beans totaled 615g before freeze-drying. More to come as I proceed with the process.

Fresh cacao beans are 60-70% water (Hart, 2019), so I am guessing that I’ll end up with around 215g of raw material after freeze-drying. Of that mass, I should lose half of it right away during fat extraction, plus another few percent in alkaloids and polyphenols (J Voigt, Wrann, Heinrichs, & Biehl, 1994). So if I do it right, let’s say I lose another 60%, leaving me with 85g of Acetone Dry Powder. About a quarter of that amount (or about 11-13% on a dry matter basis) should be made up of proteins, but only some of them are the proteins that I want (Rawel, Huschek, Sagu, & Homann, 2019). So out of the ~25g that are proteins, only about 4% are likely to be the vicilin I’m after (Rawel et al., 2019), which leaves 1g of vicilin substrate, plus if I’m lucky, a gram or so each of the enzymes.

Clearly, I need to refine this process as much as possible before I start on the multi-step protein extractions. I think it is worth cleaning up my solvent protocol again and going through the multiple acetone stages (with colder acetone) to get any polyphenols out before I start.

Works Cited

Hart, C. K. (2019). The effects of the fermentation and methods of Theobroma Cacao on quality and flavor characteristics, 110.

Janek, K., Niewienda, A., Wöstemeyer, J., & Voigt, J. (2016). The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors. Food Chemistry, 211, 320–328. https://doi.org/10.1016/j.foodchem.2016.05.033

Rawel, H. M., Huschek, G., Sagu, S. T., & Homann, T. (2019). Cocoa bean proteins-Characterization, changes and modifications due to ripening and post-harvest processing. Nutrients, 11(2). https://doi.org/10.3390/nu11020428

Voigt, J., Voigt, G., Heinrichs, H., Wrann, D., & Biehl, B. (1994). In vitro studies on the proteolytic formation of the characteristic aroma precursors of fermented cocoa seeds: The significance of endoprotease specificity. Food Chemistry, 51(1), 7–14. https://doi.org/10.1016/0308-8146(94)90040-X

Voigt, J, Wrann, D., Heinrichs, H., & Biehl, B. (1994). The proteolytic formation of essential cocoaspecific aroma precursors depends on particular chemical structures of the vicilin-class globulin of the cocoa seeds lacking in the globular storage proteins of coconuts, hazelnuts and sunflower seeds. Food Chemistry, 51(2), 197–205. https://doi.org/10.1016/0308-8146(94)90257-7

Voigt, Jürgen, Textoris-Taube, K., & Wöstemeyer, J. (2018). pH-Dependency of the proteolytic formation of cocoa- and nutty-specific aroma precursors. Food Chemistry, 255, 209–215. https://doi.org/10.1016/j.foodchem.2018.02.045