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Hop Champ Episode 6: Water Chemistry Is Bitterness
Hop Champ Episode 6: Water Chemistry Is Bitterness
In this episode we break down the exact mechanisms the cause mineral ions like Calcium, Magnesium, and particularly Calcium Sulfate to be tasted as bitter. To be clear, we are NOT talking about these ions "bringing out hop bitterness", we are clearly demonstrating that these mineral ions are themselves bitter.Subscribe to the Hop Champ Channel on Substack to access the Hop Champ IPA tasting app for Google Sheets. Click Here to Subscribe, Read More Research Notes, or Reach Out
The Notes That Built Ep 6 Are Below:
One. Do minerals added to the water chemistry of beer taste bitter themselves?
Yes. We need to be very clear and upfront about this; the calcium, magnesium, chloride, and sulfate that are added to any beer contributes to the total bitterness tasted in that beer through a very specific and well defined mechanism. There are many different cell types in the human palate that calcium and magnesium interact with to produce a wide variety of effect on the flavor and mouthfeel of a beer…and one of the cell types these minerals interact with is a taste receptor cell which only detects minerals and only sending sends bitter taste signals to the brain.
When two complex systems interact the way that they do when we taste a beer (the beer is one complex system and you the most complex system we know about), linear effects don’t exist, just general trends and increased probabilities. When we add calcium sulfate, for example, to beer, that addition does many things and one of those things is to contribute a significant increase in bitterness to the beer, but not every molecule of calcium sulfate contributes to this bitterness, just a higher amount than calcium that isn’t bonded to sulfate.
Two. Why do we taste these minerals, and the ionic compounds that contain them, as bitter?
In previous episodes of this podcast, particularly episodes four and five, we discussed some of the specialized taste perception cells on the human palate. These receptor cells are the very first stage of how we perceive taste. Generally speaking, taste begins with these receptor cells, and we have many types of them, and when those receptor cells are activated they send signals through nerve pathways, like the trigeminal nerve and the vagus nerve, to brain structures that process those signals, typically a group a neurons called the Gustatory Cortex processes the signals first before sending them to very specific places in brain structures like the Amygdala and the Hypothalamus. We taste bitterness through a specific group of receptor cells called the TAS2R group of taste receptor cells.
There are 26 varieties of these bitter taste perception cells that we know about right now, with the 26th one only recently being identified by researchers. Some of these bitter receptor cells can interact with a wide variety of bitter compounds and we see this broad interaction with many of the bitter components in hops. In the case of the water chemistry components that we’re talking about today like magnesium chloride, magnesium sulfate, calcium chloride, and calcium sulfate, which are considered ionic compounds so that’s how we’ll refer to them, we have a well documented account of a very specialized TAS2R receptor named TAS2R7 that is very precisely tuned to respond to these ionic compounds like magnesium and calcium, along with other compounds that are much less abundant in beer so we don’t have to worry about them in this discussion.
TAS2R7 is a G protein-coupled taste receptor.. Like all of G Protein-Coupled receptors, the molecules that it is specifically calibrated to respond to interact with specific amino acid residues on the receptor cell and that triggers a breakdown of the G protein into subunits, and those subunits initiate the complex signaling pathway to the brain letting it know that we’re tasting something bitter.
So, what causes the TAS2R7 receptor to send a signal that we’re tasting something bitter when it interacts with one of the minerals that we’re examining today?
A few definitions will be useful as we explore this:
Ionic Compound: is a molecule made from two or more molecules whose charges balance each other out and make the total charge of the ionic compound neutral. Ionic compounds are made up of cations and anions which are balanced to achieve this neutral charge.
cation is a positively charged ion. Calcium and Magnesium.
anion is a negatively charged ion Sulfate and Chloride.
An Ionic Compound is made up of Cations and Anions
* TAS2R7 is activated by divalent and trivalent cations, including zinc, calcium, magnesium, copper, manganese, and aluminum. Divalent and Trivalent simply refer to the specific charge of the ion. Divalent has a charge of +2, and Trivalent has a charge of +3, and these charges play a key role in how ionic compounds we’re examining are able to interact with the TAS2R7 receptor cell. These positive charges don’t just have to be positive, they have to be strong enough; Potassium, with a charge of +1, won’t interact with TAS2R7 because its charge does not cross the required energy threshold.
* Molecular Interactions: Molecular modeling suggests that the negatively charged residues in the TAS2R7, specifically H94 (histidine at position 94) and E264 (glutamate at position 264) are key to the receptor's interaction with metal ions.
* These residues are thought to interact with the metal ions through strong electrostatic interactions, meaning the interaction between positive and negative charges.
Three. What Does Our Research Cluster Say About The Effect Magnesium Has On Bitterness?
We can start with magnesium, because it’s the very straightforward example of mineral bitterness. Our research cluster shows that magnesium sulfate and magnesium chloride interact with the TAS2R7 taste receptor very significantly and cause the receptor cell to send a signal of bitter taste perception to our brain. With magnesium sulfate and magnesium chloride; the magnesium causes the response of the taste receptor cell and the anion that is paired with it, the sulfate or chloride, changes how much magnesium is required to cause a particular level of activation of the receptor cell, these studies measured two typical activation levels of the receptor cells, fifty percent activation and the maximum possible activation. The presence of sulfate increased the amount of magnesium needed to activate the TAS2R7 receptor cell compared to chloride ions. Which is interesting and something we’ll dig a little deeper into when we’re discussing calcium. So the takeaways from the research on magnesium is; anytime we’re adding magnesium to a water profile we’re increasing the bitterness of the beer. And the chloride and sulfate ions change the bitter perception of that magnesium. Obviously, when we’re talking about beer and not isolated lab experiments we’re talking about a liquid that contains a blend of chloride and sulfate ions which will result in a unique modification of magnesium’s ability to activate the TAS2R7 receptor cell.
Four. How does Calcium, in the form of Calcium Chloride, Calcium Sulfate, and ionic Calcium increase bitterness in beer?
Calcium has a more complex interaction with TAS2R7 taste receptor cells than what was observed when magnesium was tested. In our research cluster a laboratory that was studying the way minerals interact with the TAS2R7 receptor explored the interaction of calcium with the receptor cell and were able to say that the presence of calcium increases the overall activity of this receptor cell in response to any other compound that it detects. They were able to logically infer that calcium itself is activating this receptor cell.
They had to make this an inference, a logical hypothesis based on what they measured but not an official result, because the experiment didn’t test ionic calcium, or calcium not paired with sulfate or chloride, by itself, just ionic compounds paired with chlorides or sulfates. The research tested the ionic compound calcium chloride, which is very useful to us, and the researchers also used a solution containing un-paired, “ionic” calcium ions to dissolve all the compounds it was testing. So, this examination of calcium chloride took place in a solution that had calcium ions added to it before any of the tested compounds were dissolved into it, which we’ll call Free Calcium in this discussion because it was not added as an ionic compound, was the relationship that the led the researchers to make the logical hypothesis that calcium itself activates the TAS2R7.
All of these minerals were tested by dissolving them into a solution and adding them to a receptor cell that had been isolated by the laboratory. It’s important to note that ionic compounds dissociate when added to water. Full Dissociation means that when calcium chloride is added to water the ionic compound, the bond between the calcium cation and chloride anions breaks and the calcium and the chloride are dissolved into the water as individual ions. That means that when these experimenters are examining the effects of calcium chloride, they are essentially examining the effect of free calcium ions on the TAS2R7 taste receptor in the presence of Free chloride ions. This is important because it lends an additional layer of credibility to these researchers presenting the opinion that calcium itself is capable of activating the TAS2R7 receptor cell even though they did not include a direct experimental interaction of calcium and the receptor cell in their experiment.
As for the test of Calcium chloride itself, calcium chloride doesn’t have the reputation for bitterness that calcium sulfate has in the brewhouse but was documented in this laboratory research as activating this bitter taste perception cell. This finding actually aligns with numerous studies that examined the effect of these compounds on the taste of water using sensory panels, where those panels seem to universally judge water containing calcium chloride as bitter compared to water without it.
When our research cluster examined Calcium Chloride they documented a fairly complex relationship between calcium chloride, calcium, and bitter taste perception.
* Calcium Influence: The presence or absence of calcium in the solution influences the responses of TAS2R7 to chloride salts. In general, the maximal responses of the TAS2R7 receptor cell are smaller when there is no calcium present in the assay solution.
* Activation of TAS2R7: Calcium chloride (CaCl2) is identified as one of the divalent salts that can activate the TAS2R7 receptor. This means that when CaCl2 is present, it triggers a response in the TAS2R7-expressing cells.
* EC50 and Potency: As discussed, EC50 is a measure of potency, with a higher EC50 indicating a lower potency. In the case of calcium chloride (CaCl2), the EC50 value is 4.70 mM in the absence of calcium in the assay solution, and 7.56 mM when calcium is present. Half-maximal effective concentration (EC₅₀) is a quantitative pharmacological measurement that represents the concentration of a compound required to produce 50% of the maximum possible activation of a receptor.
* Lower EC₅₀ → The receptor is highly sensitive to the ionic compound (i.e., less compound is needed for activation).
* Higher EC₅₀ → The receptor is less sensitive to the compound (i.e., more compound is needed to reach 50% activation).
* This difference in EC50 values indicates that calcium chloride is less potent when calcium is present in the assay solution. The decreased potency of calcium chloride when paired with free calcium combined with the research observations that showed Free Calcium increased receptor cell response to each of the other ionic compounds studied, led the researchers to conclude that calcium itself was activating the TAS2R7 receptor cell.
* Essentially, what the research pointed out was that the TAS2R7 receptor cell seems to respond to the electrical charge of the Free Calcium in the solution and the free calcium that had been released from Ionic compound calcium chloride as well, needing more of the of the calcium originating from Calcium Chloride to increase the positive charge in the solution around the receptor cell to a degree that the receptor cell would notice because the Free Calcium in the solution prior to the addition of calcium chloride had increased the positively charge ions contacting the receptor cell enough to encourage activation, and more calcium from calcium chloride was needed to increase the positive charge to a point that would stimulate an increased activity in the receptor cell, because the receptor cell isn’t just responding the a positive charge, it’s responding to an increase the total positive charge of the ions in the liquid directly touching the receptor cell’s protein. And this makes sense, this fits with the measured conclusions that the researchers were finding: The TAS2R7 is proven to respond to specific types of cations, the divalent and trivalent cations that we mentioned earlier, and calcium is a divalent cation. This conclusion is interesting because we have ionic compounds like calcium sulfate and calcium chloride which are known to be bitter, but this taste receptor isn’t responding directly to the sulfate or the chloride, as far as we can tell, just the calcium or magnesium components of these ionic compounds… and the anions of chloride or sulfate modify the environment that the calcium and magnesium are in, and that change in the environment changes to ability of the TAS2R7 to be activated by the calcium and magnesium.
But before we move on, it’s important to note that this insight about Free Calcium and calcium chloride is a logical conclusion reached by the researchers and not something they designed the experiment to measure directly. This is common, for better or for worse, in certain types of chemistry experiments where the experiment itself is really tightly defined and any interesting phenomenon that the lab notices gets scheduled for exploration in the next experiment instead of extending the current experiment. It’s not common for a single experiment designed for research publication to see a phenomenon and change the rigorous framework of their experiment that they’ve already spent a lot of time and effort getting funded in order to perform that further exploration. It’s generally easier to note the phenomenon they witnessed, work it out to a logical conclusion, present that conclusion as a hypothesis, and then go research that interesting thing if they’re able to fund the next experiment.
So what we know for sure is that calcium chloride absolutely activates the TAS2R7 receptor cell and increases the total bitterness of beer. Calcium itself, Free Calcium, as we’ve been calling it, increases the total possible activation of the TAS2R7 bitter receptor cell in response to any magnesium used in the beer by increasing the overall activity of the taste receptor cell.. And that the researchers reached a very logical conclusion that calcium itself directly activates this bitter receptor cell even though that specific test was not built into their experiment.
Five. How does what we’ve talked about explain why Calcium Sulfate is widely recognized as bitter.
Sensory Panel Detection: Calcium sulfate has been consistently and formally identified as bitter by sensory panels in research studies. Most of these research studies have been focused on water quality and the effect that calcium sulfate has on drinking water.
Examining our researcher cluster, we can start to construct the framework that accurately describes how calcium sulfate contributes to the bitterness of beer. The foundational idea that we need to understand the way that calcium sulfate tastes bitter is that when calcium is attached to a sulfate molecule, that sulfate molecule limits the number of places that the calcium can interact with the human palate and make that sulfate-bonded calcium much more likely to interact with the TAS2R7 bitter taste receptor cell.
Five. Does calcium sulfate remain intact within beer? How does this happen?
There are a lot of things that affect the amount of calcium sulfate that remains intact within a beer from the day it is brewed until the day it winds up in your glass. For example, some yeast strains utilize sulfate in their metabolic cycle and those yeast strains will do so at different rates, and that’s a significant variable affecting the amount of calcium sulfate that remains in a beer. Those variables are important, but today we’re keeping our focus on the set of interactions that allow any calcium sulfate that’s left in the beer when it’s packaged and poured to contribute bitterness to that beer. The first thing to understand about how this happens is that a significant amount of calcium sulfate that we add to beer tends to stay calcium sulfate instead of splitting into calcium and sulfate.
When calcium sulfate is added to brewing water, in the form of gypsum, we’re adding a crystallized form of calcium sulfate, which is bonded together and breaks apart in the water resulting in calcium and sulfate ions being suspended in the water. These calcium and sulfate ions are still attracted to one another because of their positive and negative charges, which in the case of calcium and sulfate are equal, with calcium being a divalent cation with a positive charge of +2, and sulfate being a divalent anion with a divalent anion of -2. These charges can allow calcium and sulfate ions to form what are known as ion pairs. Ion pairs are temporary interactions that result in these calcium and sulfate ions becoming attracted to each other briefly. The short time that these interactions occur mean that the calcium sulfate pair remains dissolved in water while it’s an ion pair and doesn’t fall out of solution, like it would if it re-formed the crystal structure that is present in gypsum. These ion pairings are happening continuously in water and beer at a rate that is affected by a large number of factors including the number of total ions, the strength of those ions, and things like the temperature of the liquid. Sulfate is more efficient at forming these ion pairs with calcium than chloride because sulfate holds a negative charge that is equal in strength to calcium’s positive charge…and this is important because these momentary pairings can help determine the number of cell types on our palate that the sulfate-paired calcium can interact with.
The next thing that we need to understand about how calcium sulfate contributes to bitterness is the places on the palate that un-paired, or free calcium, can go when it’s not paired to sulfate in these ion parings. The short answer is that free calcium can go a ton of places and interact with many different cell types on the human palate, we won’t break down every single one of these possibilities because that would take days but we can mention a couple of those pathways that free calcium ions be used to give you a general idea.
What Cell Types On The Human Palate Can Calcium And Calcium Sulfate Interact With?
+Free Calcium: A Few Places It Can Go:
Calcium-sensing G Protein Coupled Taste Receptors: CaSR
The calcium-sensing receptor (CaSR) is a taste receptor that enhances the intensity of sweet, salty, and umami tastes. These receptor cells have been found to exist individually in mammals and also, here’s where things get interesting, these calcium sensing receptors have been in G Protein coupled taste receptors that have been previously known for detecting sweet flavors and amino acids. This particular phenomenon has been documented in the T1R3 receptor cell, which is known as one of the receptors that allows us to perceive sweetness and any amino acids in food. T1R3 was discovered to also contain this Calcium sensing mechanism as well, adding a third function to its function within our network of taste.
What is CaSR? [2, 4]
* CaSR is a G protein-coupled receptor (GPCR) that's expressed in taste cells in mammals
* It's also involved in calcium homeostasis, which is the process of regulating calcium levels in the body
How does CaSR work? [2]
* CaSR senses changes in extracellular calcium levels (Ca^{2+}) [2]
* CaSR agonists, such as γ-glutamyl peptides, glutathione, and cinacalcet, enhance the kokumi taste [1, 3]
* The CaSR-specific antagonist NPS-2143 suppresses the kokumi taste [3]
CAHLM1 Ion Channel:
Calcium homeostasis modulator 1 (CALHM1) is an ion channel that regulates calcium levels in cells and is expressed in the brain and taste buds. CALHM1 is involved in neuronal excitability and neurotransmitter release. The CALHM1 ion channel and other variations of the CALHM ion channel (these variations come as numbered versions, like CALHM3, for example) exist many places in our palate including in a large number of G-Protein taste receptors that are responsible for a detecting a wide array of flavors like sweetness, bitterness, and savory flavors as well. To interact with the CALHM group of ion channels, Free Calcium combines with ATP manufactured by cells and the calcium helps to act as a key that allows the ATP through these CALHM channels as part of the taste-signaling network which sends information about flavor to the brain.
Function [1, 3, 4]
* Regulates neuronal excitability in response to changes in extracellular calcium levels
* Mediates ATP release in taste bud cells
* Modulates electrical excitability of cortical neurons
Structure [4, 5, 6]
* A hexamer of six monomers, each with four transmembrane domains
* A pore diameter of about 14 Å
* A large pore that can accommodate fully hydrated ions or ATP molecules
Regulation [1, 7]
* Regulated by membrane voltage and extracellular calcium concentration
* Closed at resting membrane potentials but can be opened by strong depolarizations
* Reducing extracellular calcium levels increases channel open probability
Other information [4]
* CALHM1 is conserved across more than 20 species [4]
* CALHM1 has structural features that are similar to connexins and pannexins [1, 7]
* CALHM1 has been identified as a possible modifier of the age of onset of Alzheimer disease [4]
Some calcium ion channels on the human palate are voltage-gated. (CaV channels).
Explanation
* CaV channels These channels open when the membrane depolarizes, allowing calcium ions to flow into the cell. CaV channels are involved in synaptic transmission, which is the first step in sending a signal between neurons.
CALHM channels These channels are also voltage-gated and are found in the taste buds. They are permeable to large substances like ATP and may play a role in how tastes are transmitted to the brain.
CaV channels are classified as either high voltage-activated or low voltage-activated. The type of channel determines how much voltage is required to open it.
Because these channels respond to the charge of the calcium ion, anything that the calcium ion is attached to will change the charge that is detected by the ion channels. Calcium being paired with something else also effects its ability to interact with some of these ion channels, because some of them are built for calcium to flow through attached to other things like ATP, which is a cellular energy source, or certain neurotransmitters, many ion channels are built for Free, unbonded. calcium, and not ion pairs.
And this is how calcium sulfate contributes bitterness in beer. Calcium that is paired to sulfate forms a pairing with neutral charge. Like we mentioned before, calcium is a divalent cation and sulfate is a divalent anion, balancing each other;’s charges out. This neutral charge prevents the ion channels from detecting the calcium and opening. The size of the bonded ion pairing also prevents interaction between calcium sulfate pairings and ion channels on our palate. This closing a lot of paths for calcium to interact with our palate leaves the calcium portion of all the calcium sulfate pairings present in our beer to interact with the TAS2R7 bitter taste receptor cell. The TAS2R7 receptor can still interact with calcium or magnesium if they’re paired to sulfate groups, and any Free, unbonded calcium in the beer can also increase the activity of this bitter receptor cell if it reaches the TAS2R7 receptor before it gets picked up in an ion channel.
To put this in context. The calcium that we add to beer using only calcium chloride is more likely to show up as free calcium. It is highly likely to be able to interact with both ion channels or the TAS2R7 bitter taste receptor, depending on the exact physical location that it ends up on our palate. Any calcium that we add to beer using calcium sulfate can is more likely to engage in this ion pairing and have access fewer cell types to interact with, making it more likely to interact with the TAS2R7 bitter taste receptor, which leads to a significant increase in the bitterness of the beer. Calcium sulfate has a much more direct effect on bitterness than the broad effects of free, unbonded, ionic calcium because the sulfate-included ion pairing’s neutral charge and higher frequency of the ionic pairing restricts the number of cell types that calcium sulfate can interact with on the human palate. This different effects of broad acting calcium and more direct effects of calcium sulfate ion pairings are really important to keep in mind, especially when we’re building water profiles in a modern brewhouse that are based on historical water profiles from natural aquifers, because those natural aquifers have numerous environmental factors at work that can affect the probability of the ion pairing of calcium and sulfate.
The bitterness contributed by calcium sulfate is still subject to all the complexity we’ve discussed in that past couple of episodes around signaling pathways in the nervous system and signal processing structures in the brain. But the research that we’ve covered today demonstrates a clear description of the way that calcium sulfate contributes to the bitterness of a beer. Ion pairing directs a higher portion of calcium towards the TAS2R7 taste receptor cell, where even though the ion pairing with sulfate increases the total amount of calcium needed to activate the cell, the receptor can be activated more strongly because of the high amount of calcium that less available to other perception pathways of the human palate and more available to the TAS2R7 receptor cell.
Six. How is the bitterness of the ionic compounds different from the bitterness contributed by hops?
Generally speaking, the signal itself is the same, but the specific cells sending that bitterness signal are different.
* TAS2R1 interacts with hop-derived compounds. Specifically, the bitter taste of hop-derived compounds is mediated by TAS2R1, TAS2R14, and TAS2R40.
* TAS2R10 is a significant receptor for bitter compounds, interacting with prenylflavonoids such as xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin, 6-geranylnaringenin, and desmethylxanthohumol.
* Xanthohumol is considered the most bitter molecule because it interacts with all three receptors, TAS2R10, TAS2R14, and TAS2R46.
* Isoxanthohumol shows stable interaction with the key residue Ser85 of the TAS2R10 receptor and has stable contacts with other residues, which helps to stabilize the ligand.
* TAS2R14 is also a key receptor for bitter compounds, known to interact with a broad range of molecules and is considered "promiscuous". It interacts with:
* prenylflavonoids such as xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin, 6-geranylnaringenin, and desmethylxanthohumol.
* Isoadhumulone, 8-prenylnaringenin, and xanthohumol exhibit the best performance with this receptor, followed by isoxanthohumol and lupulone.
* The receptor also interacts with cis-isohumulone, cis-isocohumulone, cis-isoadhumulone, trans-isohumulone, trans-isocohumulone, and trans-isoadhumulone.
* Flufenamic acid is a known activator of TAS2R14 and the hop compounds are thought to behave similarly, binding in the same position and with high affinity.
So what we’ve seen today is that the bitterness contributed to beer by ionic compounds containing calcium and magnesium is a simple proof that the total bitterness of beer is constructed from a network of receptor cells interacting with different compounds and sending signals to our brains for processing.
This is why I believe that we have to become much more invested in measuring flavors like bitterness through sensory panels. This is why I’m advocating for Sweet-Bitter Scoring as a more relevant way to quantify the total bitterness in beer than the methods that we currently use. We need to capture the experience of people in a much more relevant and actionable way than many of the current sensory methods currently used. I also would like to point out that this doesn’t mean turning everyone into super-tasters or highly trained sensory experts, SBR Scoring provides a simple methodology for getting an average person to focus on their sensory experience to a reasonable degree, because we need to consider the experience that our guests are most likely to have, not the maximum possible effect on a trained palate in highly specialized environment, although, of course, highly trained sensory professionals hold a very important place in establishing and grounding these sensory techniques. The point is that to truly know and understand the bitter taste of beer we have to focus on the human experience of that bitterness because it’s currently the most effective way to account for all the complexity of perception and processing that arises from a diverse population of diverse molecules interacting with a diversified network of receptor cells.
Closing.
And that’s our episode. Calcium and Magnesium have a lot of effects on the way we’re able to notice flavors in beer. One of those many ways is that they increase the total bitterness in beer by interacting with the TAS2R7 taste receptor cell and if we really want to understand the bitterness in beer, then we’re going to have to come to a much more inclusive understanding of all the places that bitterness in beer emerges.
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By Ian CarletonHop Champ Episode 6: Water Chemistry Is Bitterness
In this episode we break down the exact mechanisms the cause mineral ions like Calcium, Magnesium, and particularly Calcium Sulfate to be tasted as bitter. To be clear, we are NOT talking about these ions "bringing out hop bitterness", we are clearly demonstrating that these mineral ions are themselves bitter.Subscribe to the Hop Champ Channel on Substack to access the Hop Champ IPA tasting app for Google Sheets. Click Here to Subscribe, Read More Research Notes, or Reach Out
The Notes That Built Ep 6 Are Below:
One. Do minerals added to the water chemistry of beer taste bitter themselves?
Yes. We need to be very clear and upfront about this; the calcium, magnesium, chloride, and sulfate that are added to any beer contributes to the total bitterness tasted in that beer through a very specific and well defined mechanism. There are many different cell types in the human palate that calcium and magnesium interact with to produce a wide variety of effect on the flavor and mouthfeel of a beer…and one of the cell types these minerals interact with is a taste receptor cell which only detects minerals and only sending sends bitter taste signals to the brain.
When two complex systems interact the way that they do when we taste a beer (the beer is one complex system and you the most complex system we know about), linear effects don’t exist, just general trends and increased probabilities. When we add calcium sulfate, for example, to beer, that addition does many things and one of those things is to contribute a significant increase in bitterness to the beer, but not every molecule of calcium sulfate contributes to this bitterness, just a higher amount than calcium that isn’t bonded to sulfate.
Two. Why do we taste these minerals, and the ionic compounds that contain them, as bitter?
In previous episodes of this podcast, particularly episodes four and five, we discussed some of the specialized taste perception cells on the human palate. These receptor cells are the very first stage of how we perceive taste. Generally speaking, taste begins with these receptor cells, and we have many types of them, and when those receptor cells are activated they send signals through nerve pathways, like the trigeminal nerve and the vagus nerve, to brain structures that process those signals, typically a group a neurons called the Gustatory Cortex processes the signals first before sending them to very specific places in brain structures like the Amygdala and the Hypothalamus. We taste bitterness through a specific group of receptor cells called the TAS2R group of taste receptor cells.
There are 26 varieties of these bitter taste perception cells that we know about right now, with the 26th one only recently being identified by researchers. Some of these bitter receptor cells can interact with a wide variety of bitter compounds and we see this broad interaction with many of the bitter components in hops. In the case of the water chemistry components that we’re talking about today like magnesium chloride, magnesium sulfate, calcium chloride, and calcium sulfate, which are considered ionic compounds so that’s how we’ll refer to them, we have a well documented account of a very specialized TAS2R receptor named TAS2R7 that is very precisely tuned to respond to these ionic compounds like magnesium and calcium, along with other compounds that are much less abundant in beer so we don’t have to worry about them in this discussion.
TAS2R7 is a G protein-coupled taste receptor.. Like all of G Protein-Coupled receptors, the molecules that it is specifically calibrated to respond to interact with specific amino acid residues on the receptor cell and that triggers a breakdown of the G protein into subunits, and those subunits initiate the complex signaling pathway to the brain letting it know that we’re tasting something bitter.
So, what causes the TAS2R7 receptor to send a signal that we’re tasting something bitter when it interacts with one of the minerals that we’re examining today?
A few definitions will be useful as we explore this:
Ionic Compound: is a molecule made from two or more molecules whose charges balance each other out and make the total charge of the ionic compound neutral. Ionic compounds are made up of cations and anions which are balanced to achieve this neutral charge.
cation is a positively charged ion. Calcium and Magnesium.
anion is a negatively charged ion Sulfate and Chloride.
An Ionic Compound is made up of Cations and Anions
* TAS2R7 is activated by divalent and trivalent cations, including zinc, calcium, magnesium, copper, manganese, and aluminum. Divalent and Trivalent simply refer to the specific charge of the ion. Divalent has a charge of +2, and Trivalent has a charge of +3, and these charges play a key role in how ionic compounds we’re examining are able to interact with the TAS2R7 receptor cell. These positive charges don’t just have to be positive, they have to be strong enough; Potassium, with a charge of +1, won’t interact with TAS2R7 because its charge does not cross the required energy threshold.
* Molecular Interactions: Molecular modeling suggests that the negatively charged residues in the TAS2R7, specifically H94 (histidine at position 94) and E264 (glutamate at position 264) are key to the receptor's interaction with metal ions.
* These residues are thought to interact with the metal ions through strong electrostatic interactions, meaning the interaction between positive and negative charges.
Three. What Does Our Research Cluster Say About The Effect Magnesium Has On Bitterness?
We can start with magnesium, because it’s the very straightforward example of mineral bitterness. Our research cluster shows that magnesium sulfate and magnesium chloride interact with the TAS2R7 taste receptor very significantly and cause the receptor cell to send a signal of bitter taste perception to our brain. With magnesium sulfate and magnesium chloride; the magnesium causes the response of the taste receptor cell and the anion that is paired with it, the sulfate or chloride, changes how much magnesium is required to cause a particular level of activation of the receptor cell, these studies measured two typical activation levels of the receptor cells, fifty percent activation and the maximum possible activation. The presence of sulfate increased the amount of magnesium needed to activate the TAS2R7 receptor cell compared to chloride ions. Which is interesting and something we’ll dig a little deeper into when we’re discussing calcium. So the takeaways from the research on magnesium is; anytime we’re adding magnesium to a water profile we’re increasing the bitterness of the beer. And the chloride and sulfate ions change the bitter perception of that magnesium. Obviously, when we’re talking about beer and not isolated lab experiments we’re talking about a liquid that contains a blend of chloride and sulfate ions which will result in a unique modification of magnesium’s ability to activate the TAS2R7 receptor cell.
Four. How does Calcium, in the form of Calcium Chloride, Calcium Sulfate, and ionic Calcium increase bitterness in beer?
Calcium has a more complex interaction with TAS2R7 taste receptor cells than what was observed when magnesium was tested. In our research cluster a laboratory that was studying the way minerals interact with the TAS2R7 receptor explored the interaction of calcium with the receptor cell and were able to say that the presence of calcium increases the overall activity of this receptor cell in response to any other compound that it detects. They were able to logically infer that calcium itself is activating this receptor cell.
They had to make this an inference, a logical hypothesis based on what they measured but not an official result, because the experiment didn’t test ionic calcium, or calcium not paired with sulfate or chloride, by itself, just ionic compounds paired with chlorides or sulfates. The research tested the ionic compound calcium chloride, which is very useful to us, and the researchers also used a solution containing un-paired, “ionic” calcium ions to dissolve all the compounds it was testing. So, this examination of calcium chloride took place in a solution that had calcium ions added to it before any of the tested compounds were dissolved into it, which we’ll call Free Calcium in this discussion because it was not added as an ionic compound, was the relationship that the led the researchers to make the logical hypothesis that calcium itself activates the TAS2R7.
All of these minerals were tested by dissolving them into a solution and adding them to a receptor cell that had been isolated by the laboratory. It’s important to note that ionic compounds dissociate when added to water. Full Dissociation means that when calcium chloride is added to water the ionic compound, the bond between the calcium cation and chloride anions breaks and the calcium and the chloride are dissolved into the water as individual ions. That means that when these experimenters are examining the effects of calcium chloride, they are essentially examining the effect of free calcium ions on the TAS2R7 taste receptor in the presence of Free chloride ions. This is important because it lends an additional layer of credibility to these researchers presenting the opinion that calcium itself is capable of activating the TAS2R7 receptor cell even though they did not include a direct experimental interaction of calcium and the receptor cell in their experiment.
As for the test of Calcium chloride itself, calcium chloride doesn’t have the reputation for bitterness that calcium sulfate has in the brewhouse but was documented in this laboratory research as activating this bitter taste perception cell. This finding actually aligns with numerous studies that examined the effect of these compounds on the taste of water using sensory panels, where those panels seem to universally judge water containing calcium chloride as bitter compared to water without it.
When our research cluster examined Calcium Chloride they documented a fairly complex relationship between calcium chloride, calcium, and bitter taste perception.
* Calcium Influence: The presence or absence of calcium in the solution influences the responses of TAS2R7 to chloride salts. In general, the maximal responses of the TAS2R7 receptor cell are smaller when there is no calcium present in the assay solution.
* Activation of TAS2R7: Calcium chloride (CaCl2) is identified as one of the divalent salts that can activate the TAS2R7 receptor. This means that when CaCl2 is present, it triggers a response in the TAS2R7-expressing cells.
* EC50 and Potency: As discussed, EC50 is a measure of potency, with a higher EC50 indicating a lower potency. In the case of calcium chloride (CaCl2), the EC50 value is 4.70 mM in the absence of calcium in the assay solution, and 7.56 mM when calcium is present. Half-maximal effective concentration (EC₅₀) is a quantitative pharmacological measurement that represents the concentration of a compound required to produce 50% of the maximum possible activation of a receptor.
* Lower EC₅₀ → The receptor is highly sensitive to the ionic compound (i.e., less compound is needed for activation).
* Higher EC₅₀ → The receptor is less sensitive to the compound (i.e., more compound is needed to reach 50% activation).
* This difference in EC50 values indicates that calcium chloride is less potent when calcium is present in the assay solution. The decreased potency of calcium chloride when paired with free calcium combined with the research observations that showed Free Calcium increased receptor cell response to each of the other ionic compounds studied, led the researchers to conclude that calcium itself was activating the TAS2R7 receptor cell.
* Essentially, what the research pointed out was that the TAS2R7 receptor cell seems to respond to the electrical charge of the Free Calcium in the solution and the free calcium that had been released from Ionic compound calcium chloride as well, needing more of the of the calcium originating from Calcium Chloride to increase the positive charge in the solution around the receptor cell to a degree that the receptor cell would notice because the Free Calcium in the solution prior to the addition of calcium chloride had increased the positively charge ions contacting the receptor cell enough to encourage activation, and more calcium from calcium chloride was needed to increase the positive charge to a point that would stimulate an increased activity in the receptor cell, because the receptor cell isn’t just responding the a positive charge, it’s responding to an increase the total positive charge of the ions in the liquid directly touching the receptor cell’s protein. And this makes sense, this fits with the measured conclusions that the researchers were finding: The TAS2R7 is proven to respond to specific types of cations, the divalent and trivalent cations that we mentioned earlier, and calcium is a divalent cation. This conclusion is interesting because we have ionic compounds like calcium sulfate and calcium chloride which are known to be bitter, but this taste receptor isn’t responding directly to the sulfate or the chloride, as far as we can tell, just the calcium or magnesium components of these ionic compounds… and the anions of chloride or sulfate modify the environment that the calcium and magnesium are in, and that change in the environment changes to ability of the TAS2R7 to be activated by the calcium and magnesium.
But before we move on, it’s important to note that this insight about Free Calcium and calcium chloride is a logical conclusion reached by the researchers and not something they designed the experiment to measure directly. This is common, for better or for worse, in certain types of chemistry experiments where the experiment itself is really tightly defined and any interesting phenomenon that the lab notices gets scheduled for exploration in the next experiment instead of extending the current experiment. It’s not common for a single experiment designed for research publication to see a phenomenon and change the rigorous framework of their experiment that they’ve already spent a lot of time and effort getting funded in order to perform that further exploration. It’s generally easier to note the phenomenon they witnessed, work it out to a logical conclusion, present that conclusion as a hypothesis, and then go research that interesting thing if they’re able to fund the next experiment.
So what we know for sure is that calcium chloride absolutely activates the TAS2R7 receptor cell and increases the total bitterness of beer. Calcium itself, Free Calcium, as we’ve been calling it, increases the total possible activation of the TAS2R7 bitter receptor cell in response to any magnesium used in the beer by increasing the overall activity of the taste receptor cell.. And that the researchers reached a very logical conclusion that calcium itself directly activates this bitter receptor cell even though that specific test was not built into their experiment.
Five. How does what we’ve talked about explain why Calcium Sulfate is widely recognized as bitter.
Sensory Panel Detection: Calcium sulfate has been consistently and formally identified as bitter by sensory panels in research studies. Most of these research studies have been focused on water quality and the effect that calcium sulfate has on drinking water.
Examining our researcher cluster, we can start to construct the framework that accurately describes how calcium sulfate contributes to the bitterness of beer. The foundational idea that we need to understand the way that calcium sulfate tastes bitter is that when calcium is attached to a sulfate molecule, that sulfate molecule limits the number of places that the calcium can interact with the human palate and make that sulfate-bonded calcium much more likely to interact with the TAS2R7 bitter taste receptor cell.
Five. Does calcium sulfate remain intact within beer? How does this happen?
There are a lot of things that affect the amount of calcium sulfate that remains intact within a beer from the day it is brewed until the day it winds up in your glass. For example, some yeast strains utilize sulfate in their metabolic cycle and those yeast strains will do so at different rates, and that’s a significant variable affecting the amount of calcium sulfate that remains in a beer. Those variables are important, but today we’re keeping our focus on the set of interactions that allow any calcium sulfate that’s left in the beer when it’s packaged and poured to contribute bitterness to that beer. The first thing to understand about how this happens is that a significant amount of calcium sulfate that we add to beer tends to stay calcium sulfate instead of splitting into calcium and sulfate.
When calcium sulfate is added to brewing water, in the form of gypsum, we’re adding a crystallized form of calcium sulfate, which is bonded together and breaks apart in the water resulting in calcium and sulfate ions being suspended in the water. These calcium and sulfate ions are still attracted to one another because of their positive and negative charges, which in the case of calcium and sulfate are equal, with calcium being a divalent cation with a positive charge of +2, and sulfate being a divalent anion with a divalent anion of -2. These charges can allow calcium and sulfate ions to form what are known as ion pairs. Ion pairs are temporary interactions that result in these calcium and sulfate ions becoming attracted to each other briefly. The short time that these interactions occur mean that the calcium sulfate pair remains dissolved in water while it’s an ion pair and doesn’t fall out of solution, like it would if it re-formed the crystal structure that is present in gypsum. These ion pairings are happening continuously in water and beer at a rate that is affected by a large number of factors including the number of total ions, the strength of those ions, and things like the temperature of the liquid. Sulfate is more efficient at forming these ion pairs with calcium than chloride because sulfate holds a negative charge that is equal in strength to calcium’s positive charge…and this is important because these momentary pairings can help determine the number of cell types on our palate that the sulfate-paired calcium can interact with.
The next thing that we need to understand about how calcium sulfate contributes to bitterness is the places on the palate that un-paired, or free calcium, can go when it’s not paired to sulfate in these ion parings. The short answer is that free calcium can go a ton of places and interact with many different cell types on the human palate, we won’t break down every single one of these possibilities because that would take days but we can mention a couple of those pathways that free calcium ions be used to give you a general idea.
What Cell Types On The Human Palate Can Calcium And Calcium Sulfate Interact With?
+Free Calcium: A Few Places It Can Go:
Calcium-sensing G Protein Coupled Taste Receptors: CaSR
The calcium-sensing receptor (CaSR) is a taste receptor that enhances the intensity of sweet, salty, and umami tastes. These receptor cells have been found to exist individually in mammals and also, here’s where things get interesting, these calcium sensing receptors have been in G Protein coupled taste receptors that have been previously known for detecting sweet flavors and amino acids. This particular phenomenon has been documented in the T1R3 receptor cell, which is known as one of the receptors that allows us to perceive sweetness and any amino acids in food. T1R3 was discovered to also contain this Calcium sensing mechanism as well, adding a third function to its function within our network of taste.
What is CaSR? [2, 4]
* CaSR is a G protein-coupled receptor (GPCR) that's expressed in taste cells in mammals
* It's also involved in calcium homeostasis, which is the process of regulating calcium levels in the body
How does CaSR work? [2]
* CaSR senses changes in extracellular calcium levels (Ca^{2+}) [2]
* CaSR agonists, such as γ-glutamyl peptides, glutathione, and cinacalcet, enhance the kokumi taste [1, 3]
* The CaSR-specific antagonist NPS-2143 suppresses the kokumi taste [3]
CAHLM1 Ion Channel:
Calcium homeostasis modulator 1 (CALHM1) is an ion channel that regulates calcium levels in cells and is expressed in the brain and taste buds. CALHM1 is involved in neuronal excitability and neurotransmitter release. The CALHM1 ion channel and other variations of the CALHM ion channel (these variations come as numbered versions, like CALHM3, for example) exist many places in our palate including in a large number of G-Protein taste receptors that are responsible for a detecting a wide array of flavors like sweetness, bitterness, and savory flavors as well. To interact with the CALHM group of ion channels, Free Calcium combines with ATP manufactured by cells and the calcium helps to act as a key that allows the ATP through these CALHM channels as part of the taste-signaling network which sends information about flavor to the brain.
Function [1, 3, 4]
* Regulates neuronal excitability in response to changes in extracellular calcium levels
* Mediates ATP release in taste bud cells
* Modulates electrical excitability of cortical neurons
Structure [4, 5, 6]
* A hexamer of six monomers, each with four transmembrane domains
* A pore diameter of about 14 Å
* A large pore that can accommodate fully hydrated ions or ATP molecules
Regulation [1, 7]
* Regulated by membrane voltage and extracellular calcium concentration
* Closed at resting membrane potentials but can be opened by strong depolarizations
* Reducing extracellular calcium levels increases channel open probability
Other information [4]
* CALHM1 is conserved across more than 20 species [4]
* CALHM1 has structural features that are similar to connexins and pannexins [1, 7]
* CALHM1 has been identified as a possible modifier of the age of onset of Alzheimer disease [4]
Some calcium ion channels on the human palate are voltage-gated. (CaV channels).
Explanation
* CaV channels These channels open when the membrane depolarizes, allowing calcium ions to flow into the cell. CaV channels are involved in synaptic transmission, which is the first step in sending a signal between neurons.
CALHM channels These channels are also voltage-gated and are found in the taste buds. They are permeable to large substances like ATP and may play a role in how tastes are transmitted to the brain.
CaV channels are classified as either high voltage-activated or low voltage-activated. The type of channel determines how much voltage is required to open it.
Because these channels respond to the charge of the calcium ion, anything that the calcium ion is attached to will change the charge that is detected by the ion channels. Calcium being paired with something else also effects its ability to interact with some of these ion channels, because some of them are built for calcium to flow through attached to other things like ATP, which is a cellular energy source, or certain neurotransmitters, many ion channels are built for Free, unbonded. calcium, and not ion pairs.
And this is how calcium sulfate contributes bitterness in beer. Calcium that is paired to sulfate forms a pairing with neutral charge. Like we mentioned before, calcium is a divalent cation and sulfate is a divalent anion, balancing each other;’s charges out. This neutral charge prevents the ion channels from detecting the calcium and opening. The size of the bonded ion pairing also prevents interaction between calcium sulfate pairings and ion channels on our palate. This closing a lot of paths for calcium to interact with our palate leaves the calcium portion of all the calcium sulfate pairings present in our beer to interact with the TAS2R7 bitter taste receptor cell. The TAS2R7 receptor can still interact with calcium or magnesium if they’re paired to sulfate groups, and any Free, unbonded calcium in the beer can also increase the activity of this bitter receptor cell if it reaches the TAS2R7 receptor before it gets picked up in an ion channel.
To put this in context. The calcium that we add to beer using only calcium chloride is more likely to show up as free calcium. It is highly likely to be able to interact with both ion channels or the TAS2R7 bitter taste receptor, depending on the exact physical location that it ends up on our palate. Any calcium that we add to beer using calcium sulfate can is more likely to engage in this ion pairing and have access fewer cell types to interact with, making it more likely to interact with the TAS2R7 bitter taste receptor, which leads to a significant increase in the bitterness of the beer. Calcium sulfate has a much more direct effect on bitterness than the broad effects of free, unbonded, ionic calcium because the sulfate-included ion pairing’s neutral charge and higher frequency of the ionic pairing restricts the number of cell types that calcium sulfate can interact with on the human palate. This different effects of broad acting calcium and more direct effects of calcium sulfate ion pairings are really important to keep in mind, especially when we’re building water profiles in a modern brewhouse that are based on historical water profiles from natural aquifers, because those natural aquifers have numerous environmental factors at work that can affect the probability of the ion pairing of calcium and sulfate.
The bitterness contributed by calcium sulfate is still subject to all the complexity we’ve discussed in that past couple of episodes around signaling pathways in the nervous system and signal processing structures in the brain. But the research that we’ve covered today demonstrates a clear description of the way that calcium sulfate contributes to the bitterness of a beer. Ion pairing directs a higher portion of calcium towards the TAS2R7 taste receptor cell, where even though the ion pairing with sulfate increases the total amount of calcium needed to activate the cell, the receptor can be activated more strongly because of the high amount of calcium that less available to other perception pathways of the human palate and more available to the TAS2R7 receptor cell.
Six. How is the bitterness of the ionic compounds different from the bitterness contributed by hops?
Generally speaking, the signal itself is the same, but the specific cells sending that bitterness signal are different.
* TAS2R1 interacts with hop-derived compounds. Specifically, the bitter taste of hop-derived compounds is mediated by TAS2R1, TAS2R14, and TAS2R40.
* TAS2R10 is a significant receptor for bitter compounds, interacting with prenylflavonoids such as xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin, 6-geranylnaringenin, and desmethylxanthohumol.
* Xanthohumol is considered the most bitter molecule because it interacts with all three receptors, TAS2R10, TAS2R14, and TAS2R46.
* Isoxanthohumol shows stable interaction with the key residue Ser85 of the TAS2R10 receptor and has stable contacts with other residues, which helps to stabilize the ligand.
* TAS2R14 is also a key receptor for bitter compounds, known to interact with a broad range of molecules and is considered "promiscuous". It interacts with:
* prenylflavonoids such as xanthohumol, isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin, 6-geranylnaringenin, and desmethylxanthohumol.
* Isoadhumulone, 8-prenylnaringenin, and xanthohumol exhibit the best performance with this receptor, followed by isoxanthohumol and lupulone.
* The receptor also interacts with cis-isohumulone, cis-isocohumulone, cis-isoadhumulone, trans-isohumulone, trans-isocohumulone, and trans-isoadhumulone.
* Flufenamic acid is a known activator of TAS2R14 and the hop compounds are thought to behave similarly, binding in the same position and with high affinity.
So what we’ve seen today is that the bitterness contributed to beer by ionic compounds containing calcium and magnesium is a simple proof that the total bitterness of beer is constructed from a network of receptor cells interacting with different compounds and sending signals to our brains for processing.
This is why I believe that we have to become much more invested in measuring flavors like bitterness through sensory panels. This is why I’m advocating for Sweet-Bitter Scoring as a more relevant way to quantify the total bitterness in beer than the methods that we currently use. We need to capture the experience of people in a much more relevant and actionable way than many of the current sensory methods currently used. I also would like to point out that this doesn’t mean turning everyone into super-tasters or highly trained sensory experts, SBR Scoring provides a simple methodology for getting an average person to focus on their sensory experience to a reasonable degree, because we need to consider the experience that our guests are most likely to have, not the maximum possible effect on a trained palate in highly specialized environment, although, of course, highly trained sensory professionals hold a very important place in establishing and grounding these sensory techniques. The point is that to truly know and understand the bitter taste of beer we have to focus on the human experience of that bitterness because it’s currently the most effective way to account for all the complexity of perception and processing that arises from a diverse population of diverse molecules interacting with a diversified network of receptor cells.
Closing.
And that’s our episode. Calcium and Magnesium have a lot of effects on the way we’re able to notice flavors in beer. One of those many ways is that they increase the total bitterness in beer by interacting with the TAS2R7 taste receptor cell and if we really want to understand the bitterness in beer, then we’re going to have to come to a much more inclusive understanding of all the places that bitterness in beer emerges.
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