Is this Real chocolate? Reese's Vs. Claudio Corallo by Mass Spec Everything Proteomics

[00:00:00]Is this really chocolate? And what constitutes real chocolate? For this video, we're going to focus on the proteins.

[00:00:08]My thought is if it has seed-stored proteins from cacao, from the cocoa bean, then it is chocolate. All right, so we got the CC chocolate versus the Reese's chocolate. We're going to just cut off some from the side of the Reese's and break up some of this Sao Tome Principe chocolate and put it in the tube here.

[00:00:28]All right, so you can see the Reese's chocolate way lighter milk chocolate.

[00:00:32]All right, so the chocolate's melted in water, about 50/50, so they're like a nice chocolate slurry. You can definitely tell that the Reese's is a lot smoother.

[00:00:44]Uh and then I'm precipitating it again.

[00:00:46]So, 200 microliters into 1 mil of the acetonitrile zinc precip solution.

[00:00:52]All right, two protein fractions all cleaned up. On the left, we got the CC.

[00:00:56]On the right, we got the Reese's on the right.

[00:00:58]All right, after reduction and alkylation, you can see the protein.

[00:01:03]There's a big difference in just the reduced and alkylated intact protein prior to digestion. It just looks different. Digested.

[00:01:13]See if you can guess which one's which.

[00:01:15]All right, so diluted out formic acid, 0.2%, and then we're going to spin them out.

[00:01:20]All right, Theobroma cacao digesting chocolate. Reese's versus high-end chocolate, Zotter Cu Tavo. All right, here's the digested protein data. You can see the high-end chocolate on top has less peptides than the milk chocolate Reese's on the bottom. I'm thinking this is because of digestibility of all the milk proteins.

[00:01:43]And then also, it's probably easier to digest proteins that are highly processed. And so, my thought is that there's some sort of pre-production enzymes being used, probably before roasting the cocoa beans, uh that produces easier to digest proteins uh in the processed chocolate versus kind of this high-end like naturally authentic produced chocolate. Figure this stuff out on our own. We're going to send this through peaks database searching figure out what all these peptides are.

[00:02:11]So, we got the peaks chocolate data.

[00:02:14]Search the database and you can see here top hit is the 21 kilodalton seed storage protein from Theobroma cacao.

[00:02:22]Based on the area, you can see that the the high-end chocolate doesn't really have any milk in it. It's all zero area. So, I again, you can see that there is for sure Theobroma cacao seed storage and this vicilin protein from Theobroma cacao in the Reese's sample. I think that confirms it's chocolate. This is an investigation of flavor profiling in the cocoa beans full proteomic huge thesis paper here in the Reese's versus the high-end chocolate. This is good data.

[00:03:01]I can't confirm all these peptides with these areas. So, at least these first top peptides were all found in the Reese's sample. More peptides coming from the high-end chocolate, but the Reese's chocolate has very intense peptides which cover a majority of this 21 kilodalton seed storage protein. This is we look up this protein. This is the 21 kilodalton seed protein. You can see the cool things that we're finding that there's a signal peptide. So, usually when these are produced and exported outside the cell, the signal peptide gets chopped at position 26.

[00:03:39]We are missing 26. It's exactly. So, that piece isn't there, which means it's an expressed protein.

[00:03:49]Very interesting. And then we also know that there's a disulfide bond from 69 to 116. So, we modified with our digestion protocol, so we should be able to tell that disulfide bond is there, 69 to 116 in the software. Boom, maps it.

[00:04:05]Cuz the propenamide, that propenamide is from 69 to 116. We broke that disulfide bond and we mapped it with the propenamide, that acrylamide modification from the procedure. Just to confirm, yes, the signal peptide is completely removed, removed when the 21 kilodalton seed storage protein is naturally expressed inside the cocoa bean. Research from the Nestlé Corporation reveals that there's a C-terminal cut, so the mature is actually nine amino acid residues shorter. So, you're cutting off some of the end of the protein as well. And then, you can see in my data, we are missing some of that end.

[00:04:45]We're not seeing this mapping either. I don't know where that little piece went, but it does have a lot of cleavage sites, so maybe it's all chopped up. I don't know. But, we're missing two regions, and one of them is this tiny piece of the end, which it says that is part of the mature dual end trimming. Is the standard behavior for plant seed storage proteins. Very interesting, guys.

[00:05:03]All right, here it is. This is the Reese's chocolate and the high-end chocolate processed against chocolate only database. This is all of UniProt, like 50,000 chocolate proteins only. You can see at the top, we got that 21 kilodalton seed storage protein and the vicilin A mapped at high intensity for both Reese's and the high-end chocolate.

[00:05:27]But then, the most interesting thing is that you can see this 2S albumin seed storage protein. We got five peptides and a good amount of coverage in the high-end chocolate, and we're missing it completely in the Reese's. So, the question is, where is that seed storage protein?

[00:05:45]All right, so I asked the Claude AI this time that question. Why is there less 2S albumin, basically none, in the Reese's and they're saying it's due to a preprocessing enzyme. So, if you allow naturally cocoa beans to ferment after you harvest them prior to roasting, the natural endopeptidases and break up this 2S albumin seed storage protein. However, we are doing that naturally in the high-end chocolate and then it's being roasted which then produces a lot of free amino acids for the Maillard reaction in chemistry which gives you all of your flavor. There's probably some pre-roast enzymatic treatment to try to like like normalize all the different batches of cocoa beans that large producers like Reese's are taking in. So, they will probably further push the proteolysis using natural fermentation and potentially the combination of Flavorzyme or Alcalase synthetic enzyme. These are exogenous commercial proteases that are used in the food sector to further process and help enhance flavor and kind of normalize mass-produced protein strains. And so, I'm guessing that there is some sort of pre-roast processing at the industrial scale that's going on in the Reese's chocolate and that accounts for the easier digestibility and the lack of the 2S albumin storage protein.