Key Factors Influencing Enzymes

Key Factors Influencing Enzymes

A number of enzymes we describe by name in this thread are really groups of enzymes. Because of the specificity of any given enzyme, they may only act on a single bond in any given substrate. For example, Beta-glucanase as a generalization may refer to any or all of Endo-B-1.4-glucanase, Endo-B-1.3-glucanase, exo-B-glucanase, or even B-glucan solubilase. Each of these individual enzymes has a very specific action, however they are all under the umbrella of acting on beta-glucans (same substrate) in some way.

Supplemental enzymes can be derived from alternate sources, such as bacteria or fungi. In this way it is possible to derive usable quantities of more temperature stable enzymes, allowing alternate processing methods and increased reaction speeds.

Recently I had a colleague from France ask me for some advice on a new Witbier recipe he was looking to move from trial batch to production volume. Specific concerns centered on processing times and extraction efficiency in the lauter. After a brief exchange of emails he proceeded with confidence, and my most recent update is that the wort is fermenting nicely.

When deciding the mash procedure, it is paramount to consider the raw ingredient composition. Wheat is one ingredient that can benefit from a targeted mash schedule, and in this specific example, I offered my colleague advice to help direct flavor production, maximize efficiency, and insure against processing difficulties.  

Previously we mentioned the importance of enzyme shape and a bit about how they work. In order to capitalize on the benefits of enzymatic activity it is important to understand some key influencing factors, and how to control them.

Temperature is by far the most impactful to enzyme activity. Too low and reaction activity will take extended periods. Too high, and you risk denaturing (reshaping the structure) of the enzyme and rendering it useless. The purpose of a single infusion mash is to balance all required enzymatic activity at one temperature, providing for a simple processing experience. More complex mash techniques make use of temperature rests targeting specific enzymatic activity.

pH is the second most influential factor in enzymatic activity. It can change the shape and function of proteins, which impacts the ability to catalyse reactions. As with temperature, often an optimal range can be identified and targeted. Acidification of the mash is commonly achieved through a variety of means. Food grade lactic or phosphoric acid are easy, but more traditional options such as acidulated malt, sour mash, or even roast barley/plack patent malt have been used for over a century.

Let us take our hand in glove example from the previous post. Using a standard leather glove as our enzyme and our hand as the substrate, we have a practical example of temperature influence. At room temperature your hand and the glove can both manipulate contact to an ideal result. If you place that glove in the freezer for an hour first, your hand will not likely fit into the glove, based on its rigid shape. If you put that glove on the kitchen stove, it will burn and your glove will be permanently denatured. For pH, we will consider the threaded stitching of the glove. Too much and your fingers will not fit in, too little and it will not hold your hand. While the stitching is important and critical to the situation, it is less of a factor than the temperature of the glove, until you reach extremes.

At 99*F (37*C) the enzymes beta-glucanase and phytase have started to become active, and by 104*F (40*C) Beta-amylase, and proteolytic enzymes have also activated. The activity of these enzymes will increase through an optimal range, eventually denaturing before the kettle.

Beta-glucanase provides a major service in accessing the starch molecules because, as it’s ase name implies, it acts upon beta-glucans. In our last post we explained the cellular structure of starch, which contains two layers of beta glucans surrounding the middle lamella, and those layers of glucans were in turn coated with a “net” of pentosans. These pentosans have organic acids, such as ferulic acid, bound to them. Xylanases can degrade organic acids and pentosans, exposing the more rigid structure of the beta glucans to be acted on by the beta-glucanases. This is important for starch access/conversion. Ferulic acid is a precursor to 4-vinyl guaiacol, a “clove” flavor desirable in specific beers using certain yeast strains, such as the Witbier mentioned.

Phytase will influence the acidity of the mash, although it should not be relied upon to reach optimal pH due to the time required. It catalyses pythin, an organophosphate, into pythic acid, and insoluble magnesium, and calcium phosphates. Pythic acid content of barley averages about 1%, declining to about 0.78% in malted barley. A rest at 99-131*F (37-55*C) could have been an evolution of brewing technique required by high percentages of lightly kilned malts (and water profiles) that resulted in higher than ideal mash pH and weak enzymatic activity. Other more practical means of acidification can be used today such as acidulated malt, food grade lactic or phosphoric acid, or even 0.5% roasted barley/black patent malt in the grist.

Proteolytic enzymes, proteinase and peptidase, are responsible for acting on proteins by breaking large molecules into smaller amino chains, and then cleaving the ends off of those amino chains, respectively. Proteinase must be delicately balanced to ensure no haze forming proteins are left in the finished product, but not so few proteins to negatively affect foam stability. Peptidase plays a very important role in “freeing” amino acids (Free Amino Nitrogen, or FAN) and small peptides which will be required by the yeast during fermentation.

By suggesting a short (10min) mash rest at 113-122*F (45-50*C) we are attempting to unlock the ferulic acid content by offering a balance between the enzymes forementioned. Since acidity can be adjusted quickly and economically and because haze formation is not a primary concern in Witbier, we are really focusing on the “gum conversion” of xylanase and beta-glucanase to free the ferulic acid from the pentosan layer and expose starch molecules. Rest time is very important as extended degradation by proteolytic enzymes (proteinase and peptidase) can lead to a thin and watery mouthfeel, and foam retention issues.

A slightly longer rest of 30 mins is suggested at 144*F (62*C), which is approaching an optimal range for beta-amylase activity, and has activated alpha-amylase. Here the two can begin the saccharification process together with Alpha-amylase braking amylose and amylopectin chains into 7-12 molecule glucose “residues”. Beta-amylase then splits maltose (two conjoined glucose molecules) from these residues at the ends of the chain, eventually leaving only “limit dextrinases” (the leftovers of amylopectin after being acted upon by alpha and beta amylase). Any “extra” glucose molecules from the amylase activity may stay as maltotriose (three glucose molecules), or may be left as a single glucose molecule during the saccharification process.

Heat is to be applied gently over the next 30 mins raising the temperature to 176*F (80*C), which will allow a shift through peak beta amylase activity to peak alpha amylase activity, and then denaturing to stop all enzymatic activity before lautering. This should lead to a highly fermentable wort composed primarily of maltose, but also containing elevated levels of ferulic acid for later development into 4-vinyl guaiacol “clove” character.

In addition to the aforementioned suggestions, I also recommended the use of empty rice hulls in the mash. This is a common aid, and helps to “lighten” the lauter bed when using ingredients that have a tendency to compact during run off. The rice hulls offer no enzymatic advantage, but rather aim to improve physical extraction.

While targeting optimal enzymatic conditions can significantly improve conversion, efficiency is not gained without proper extraction. Extended rests in the cytolytic and proteolytic range can lead to a physical degradation of the lauter bed, potentially causing physical extraction problems. Supplemental enzymes can be obtained for similar purposes as the low temperature rests, but must be balanced with the same potential concerns. If proper consideration is taken, increased real extract, shorter rests, and improved attenuation are quite possible.

Hopefully this information was beneficial to my friend in his quest for a new Witbier. I will circle back with him in the coming weeks to see how the beer developed and what exact procedures he opted for. Ironically, the same day I was responding to this colleague, another reached out with questions on alternative ingredients. Since I was now deeply involved in the thought process (AKA 6-pack), I was clearly ready to offer some more advice.

Somewhat analogous to the wheat we have just discussed, rice and maize are two more common brewing ingredients that bring special considerations. Sure you can throw in a few bags of flaked products and cross your fingers, but what if you want to go to the extreme?

Unfortunately to gelatinize and then liquify rice or maize, the temperature must be above the activity level of barley based amylases. Basically, you have to boil the rice or maize in order to fully hydrate the starch contents, and that denatures enzymes necessary for starch conversion. This sounds complicated...

Traditional methods would suggest proportional mixing of barley mashes and adjunct mashes to aid in the access and break down of the adjunct starch. A small percentage of barley, mashed with the adjunct at 122*F (50*C), can be rested for a short period then heated for gelatinization and liquefaction. It is typically boiled for the latter, and to aid in raising the combined mash temperature. The low initial adjunct mash temperature allows cytolytic and proteolytic enzymes to work on the adjunct, and the temperature rise fully hydrates and separates the starches for later access by amylases. The remainder of the barley is mashed separately at the same 122*F (50*C), and rested for the appropriate time. The boiled adjunct is then blended back to the barley mash, very carefully, as to reach each desired temperature rest step. This is where the amylases can perform saccharification, and proteolytic enzymes can contribute to the FAN content. Adjuncts can reduce the amount of overall FAN available for yeast, so this is an important consideration when using large quantities of adjuncts.

Modern times bring some new processing aids. Today, it is possible to use added enzymes to avoid the cereal cooking process altogether. Certain blends may allow for liquefaction below the normal gelatinization temperature, and more consistency and flexibility when dealing with adjunct blends or unusual ingredients. Increased extract, reduced conversion times, and increased attenuation are all quite possible if the proper concerns are understood. Although we discussed three of the most common adjuncts today, there’s a countless number of others that may pop up in future posts!

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