6
Mustard—Fiercely Yours
The cups and the pots and the pans are to the kitchen what beakers, flasks and test tubes are to the chemistry laboratory. Every time you cook, dozens, hundreds or even thousands of reactions take place, more or less simultaneously. Some of these reactions are by nature enzymatic, where enzymes play a vital role either before, during or even after cooking. Enzymes are a subgroup among proteins that function as catalysts, the worker molecules among proteins that facilitate various reactions that otherwise would not have occurred or that would have required much heating. Since many enzymes in plant and animal cells are still operational in non-heated food materials, vegetables, fruits, meat, and even the wheat flour that we buy from grocery stores are full of different types of active enzymes.
A very familiar enzymatic reaction to most of us is the formation of strong, pungent substances from garlic and onion that can make us cry. Whole onion and garlic are very mild in flavor with no detectable pungency or kick. The pungency emerges if the plant cells are crushed and enzymes come in contact with certain compounds elsewhere in the cell. The unharmed cell has an organized structure where enzymes and their target molecules are isolated from each other, but when we start cutting, bruising, or chewing the onion, at the same time wreaking havoc in the cells, the enzymes and their target molecules come into contact with each other and enzymatic reactions take place. Since many insects and animals find this pungency unpleasant, these reactions function as a defense mechanism for the plant. In addition to garlic and onions, such reactions to produce pungent compounds play a role in a number of plants such as mustard, wasabi, horseradish, and many kale plants such as rucola.
^Proteins (center) are long chains of amino acids. They form spirals that bundle up to form three-dimensional structures. The icons indicate how we have chosen to display the various classes of proteins in this book.
In the kitchen, enzymatic reactions may be tricky and unpredictable, sometimes causing favorable and at other times unfavorable outcomes. But it is possible to learn to tackle them and take advantage of their potential. The first and foremost thing to remember is that enzymes are proteins, and behave as such. If they are overheated, they will denature or even coagulate, and thus lose their ability to function as enzyme. The critical point is, in many cases, between 70 and 80°C. So, if you want to prevent an enzymatic reaction, make sure to heat the food, such as by blanching (plunging in boiling water for a short while and then transferring to ice water to stop further cooking). Or you can make onions or garlic taste mellow and mild by cooking them whole without crushing or cutting them. This way the enzymatic reaction will then never take place since the enzymes and target molecules will not meet, at least not before the enzymes are deactivated by the heat. On the other hand, if you want to make use of an enzymatic process, you may have to introduce a moderate amount of heat to reach the optimum reaction temperature for the enzymes while at the same time taking care not to heat the food too much so that the enzymes are permanently deactivated.
At their best, enzymes turn chewy meat tender, i.e. when the protease enzymes during hanging or low-temperature cooking break down chewy proteins. Cured meats have never been cooked, and after a visit to the salt tub, they are left to dry; meanwhile, the enzymes get active to make the meat tender over time. Brewers make use of amylase enzymes in the mashing step of beer brewing to break down starch from cereals to sugars, which the yeast uses to produce alcohol, carbon dioxide, and flavor compounds. In Finland, amylase enzymes are in action every Christmas to make a sweet potato casserole (imelletty perunalaatikko) described in the chapter about porridge. Some enzymes in foods we want to prevent, such as the polyphenol oxidase enzyme, which turn some fruits and vegetables brown. Another enzyme that can prove really frustrating is pectin methylesterase, which under certain conditions prevents vegetables in softening during cooking. More about this rascal in the next chapter.
Proteins, and thus enzymes, are long strings of amino acids organized into spirals, folds, and kinks, which are again folded into three-dimensional bundles. These have specially shaped pockets or sites where only specific molecules, target molecules, via random encounters can fit in. Almost like a hand in a glove, Cinderella’s shoe, or a piece of a puzzle. When such a “docking” has occurred, an enzymatic reaction can take place. For example, the target molecule can be split in two as the enzyme facilitates breaking of a chemical bond. Sometimes, such reactions are described as key-in-lock reactions because the target molecule must fit into the pocket of the enzyme for a reaction to occur. If a molecule comes by that does not fit into the pocket—no docking and no reaction.
There are, however, some molecules that do fit in the pocket but no reaction occurs because they do not possess the specific bond to be broken or for some other reason. These enzyme inhibitors fit into the pocket of the enzyme molecule but no reaction occurs. Some inhibitors are rather persistent and, unlike true target molecules that leave the enzyme pocket straight after the reaction, they stay put and block the enzymatic reaction. In fact, winemakers and food manufacturers use this to their own benefit, and our pleasure. A commonly used inhibitory molecule is sulfite (HSO3−) or sulfur dioxide (SO2), which in water reacts to produce sulfite. This fits perfectly into the active site of the polyphenol oxidase (PPO) enzyme responsible for fruits and vegetables turning brown when the plant cells are broken. As this reaction pocket in the enzyme is occupied by sulfite, the browning reaction cannot take place and apricots or apples to be dried, or green grapes used in juices and wines maintain their original color.
Back to mustard. In Finland, homemade mustard is a common Christmas gift for friends and relatives, and in the weeks and months before the festive season food magazines are full of mustard recipes with that “secret” ingredient or trick to make it special. Some season their home-made mustard with cognac or other strong spirits, others rely on herbs such as tarragon. Some want mild, round, and sweet mustard and therefore add cream and honey. “Is there any science in mustard?” Tatu the chef asked as Christmas was approaching one year. Thus, we were reminded of something we had wanted to study for a long time: how do the enzymatic reaction conditions affect the development of pungency in mustard?
The key ingredients of mustard are mustard powder, water, and vinegar, and the pungency develops as result of reactions between these three. However, in the various mustard recipes these core ingredients are treated quite differently. In some recipes water is to be added cold, in others boiling hot. Some recipes ask for the vinegar to be added in the beginning whereas others instruct us to mix mustard powder and water well before adding any vinegar. As the pungency of mustard develops through an enzymatic reaction, temperature and the mixing order of ingredients should make a difference. Different procedures should result in different styles of mustard, even with the exact same ingredients.
Mustard seeds are in some respect like garlic or onion—they are not pungent at all before they are crushed. When the cells are broken in the presence of water, the enzyme myrosinase will soon encounter molecules that were originally in other parts of the cell. These target molecules, glucosinolates, are built up by a glucose backbone, an amino acid part and a side group (“tail”) that may vary. The highly volatile and very pungent sulfur-containing side group is released if the glucosinolate molecule should enter the active site of the myrosinase enzyme (see figure). This sharp pungency that we humans have learnt to enjoy also acts as a defense mechanism against insects or animals who might find it unpleasant and therefore seek more mellow snacks.
As private consumers, we don’t have the same easy access to sulfite as the food and drinks manufacturers. Perhaps it is just as well since sulfite should not be consumed in large amounts, which is also why dried fruits and white wines have disclaimers of containing traces of sulfur. An easily accessible alternative exists, however, that would give the same benefit albeit through another mechanism. The enzymatic browning blocked by sulfite is an oxidation process, and oxidations can be prevented or reversed by antioxidants. Ascorbic acid (vitamin C) abundant in the juice of citrus fruits is an efficient antioxidant, thereby the well-known trick of sprinkling lemon juice onto freshly cut fruits or avocados to prevent browning. Today we have access to an even more potent source of this antioxidant, namely vitamin C supplements. Next time you want to avoid brown fruit, dissolve a quarter of a vitamin C tablet in a little water and sprinkle it over the fruits or vegetables that would otherwise turn brown. The main mechanism of vitamin C when retarding browning is to pick up a key substance before it has time to react to produce those brown compounds that have their function in nature but are unwanted in the kitchen (it may also under some conditions display some enzyme inhibition action similar to sulfite). The outcome is however the same: fruits and vegetable maintain their fresh color. ×
This enzymatic reaction in mustard can also be perceived in other plant-based foods such as various types of cabbage, horseradish, wasabi, and radish.
The pungency from mustard is due to various glucosinolates: sinalbin from white mustard (Sinapis alba, “true mustard”) and sinigrin from black mustard seeds (Brassica nigra, which actually belongs to the cabbage family). The pungent compound released from sinalbin is rather unstable and the pungency developed from white mustard seeds fades away rather rapidly. After six minutes in pH-neutral conditions only 50% of it is left. However, if the pH is decreased from a neutral 7 to 3.5, for example by the addition of vinegar, then the half-life of the pungent compound will increase to more than 5 hours, so the pungent compounds from sinalbin are somewhat more stable in acidic conditions. Thus, adding vinegar in the beginning of the reaction would significantly help if pungency is desired in the freshly made mustard. The pungent compound released from sinigrin is much more stable and thus black mustard seeds are a better choice for mustards that are to stay hot for more than a day. Usually commercial mustard powders are made from a mixture of white and black mustard seeds.
^When cells in mustard seeds are crushed, an enzymatic reaction occurs where myrosinase enzyme (blue) cleave glucosinolate molecules (yellow) thereby liberating the sulfur-containing side group as volatile compounds giving a sharp, burning sensation.
To summarize from mustard and enzyme theory:
1. The pungency of mustard develops via an enzymatic reaction.
2. The reaction has certain minimum requirements, an active enzyme and conditions where it can meet the target molecule, among others. This can occur when cells are broken in the presence of water.
3. Reaction conditions, especially temperature and pH, have significant impacts on the enzymatic reaction and the stability of pungent molecules.
4. It is possible to inhibit enzymatic reactions by adding a suitable inhibitor that fits into the active site of the enzyme.
With these facts in mind, we started to build our experimental setup to learn more about how the theory goes together with the practicalities of making mustard. Since we were tasting very pungent products, we limited the number of samples to three different mustards, as we wanted to avoid scaring the workshop participants away from future workshops. We had to limit ourselves and picked the two questions we thought were most interesting to study:
– How does the reaction time affect the pungency?
– How does destroying the enzyme affect the pungency and overall flavor?
The three versions were based on Tatu’s basic recipe.
According to theory, the myrosinase enzyme should be deactivated by the high temperature in Version 1, resulting in a mustard with virtually no pungency. The two next versions should give different degrees of pungency, of which Version 3 should be the most pungent as the enzymes would have had more time to react and produce pungent compounds.
The ten workshop participants were, in a blind tasting, served the mustards and asked to pick the most pungent one, the least pungent one, and which one they preferred most. In addition, they were asked to verbally describe the experiences and flavors given by the different mustards.
Mustard
Ingredients per portion
150 g mustard powder (containing white and black mustard seeds)
150 g water
50 g red wine vinegar
150 g sugar
10 g salt
Procedure according to standard recipe
Heat water and vinegar together. Add sugar and salt and let it dissolve completely. Let the mixture cool down to below 70°C. Mix in the mustard powder and let the mixture rest for 5–15 minutes. Heat to a boil and transfer to glass jar.
Experiment
For our experiments the heating process was modified in the three parallels. The liquids were mixed and heated to a boil, whereby sugar and salt were dissolved into the liquid mixture. Mustard powder was then added to this mixture in three different ways:
Version 1: Mustard powder was added to the hot liquid mixture at 80°C and kept hot under cover for 15 minutes.
Version 2: The liquid mixture was cooled to 30°C, mustard powder was added and kept at this temperature under cover for 5 minutes. The enzymatic reaction was then stopped by quickly heating the mixture to a boil.
Version 3: The liquid mixture was cooled to 30°C, mustard powder was added and kept at this temperature under cover for 15 minutes. The enzymatic reaction was then stopped by quickly heating the mixture to a boil.
The panel was quite agreed in selecting the most and the least pungent mustard. Seven out of ten panel members picked Version 3, the mustard with 15 minutes incubation time with the enzyme, to be the most pungent. A few considered Version 2, the mustard with 5 minutes incubation time with the enzyme, as the most pungent, so these two mustards were perhaps not radically different in terms of pungency. The panel was unanimous in selecting Version 1, the one heated from the very beginning, to be the least pungent of all. This was verbally described as “least mustardy” with descriptions such as “beany,” “grassy,” “bitter,” and “not tasting of mustard.” They preferred the two first versions, described as “mustardy,” “balanced,” and with “good pungency/strength.”
What did we learn from this? At least, it became obvious that pungency in mustard can be significantly altered by carefully selecting the reaction conditions, time and temperature. You can prevent the development of pungency completely by heating, thus deactivating the myrosinase enzyme before it gets the opportunity to react. You can also fine tune the level of pungency by controlling the reaction time. In our case both the 5-minute and 15-minute versions were quite pungent. For a milder version, a significantly shorter time, say 30 seconds or 1 minute, might be quite sufficient before heating to a boil. Enzymes are undoubtedly sensitive, but quick if they first get the opportunity to react.