What's Mashing?

Can an understanding of enzyme activities reduce mashing times and costs?

Dear Brew Enthusiast,

Welcome to yet another episode of the Beerologist, where beer meets science.

In this weeks' article, we are looking into mashing. Specifically, I will describe a paper that focuses on two major enzymes, α-Amylase and β-Amylase, present in malted grains and responsible for starch conversion.

Mashing is a process that, due to a high-temperature cycle, comes with significant energy demand. Therefore, from a brewers' perspective, reducing energy use (and its cost) of the mashing step can have real economic and environmental benefits. However, one critical tradeoff brewers face is that changing the mash cycle or temperatures can affect the yield of fermentable sugars. Therefore, brewers would benefit from knowing the optimal conditions for their ingredients in an ideal world when deciding on their mash programme. Unfortunately, and from an analytical perspective, this is not an easy task. 

A recent paper describes a new and relatively simple method that allows brewers to measure the activity of both α-Amylase and β-Amylase proteins, the two primary enzymes responsible for converting starch to sugar. The methodology and research that underpins the researchers' claims are below. This work is interesting for two main reasons:

1. They show that by altering (shortening) the mash cycle and understanding the effects on amylase activity, more economical mash schedules can be devised at the brewery without sacrificing yields.

2. The authors show that they can carry out this work using a kit that is accessible to brewers and thus, should enable smaller craft brewers (and serious hobbyists) to do this work themselves. 

What they did

First, the authors made sure that their milled malts had a particle size similar to those used in the industry. Particle size is important as differences in the surface area affect the release of nutrients (including starch) and proteins (including Amylases) during the mash. Subsequent analyses of the malts revealed that other parameters were roughly the same (though they observed some differences). 

The authors first measured α-Amylase and β-Amylase activity throughout the mashing process. The authors took samples throughout the mashing procedure (including the ramps and mash-out stages) and, notably, did so for multiple mashing profiles. These analyses revealed an initial burst in enzyme activity in all mashing profiles, followed by a plateau (probably due to optimal activity). The authors showed that, and consistent with the literature, α-amylase is more stable (evidenced by a limited drop of activity over time) than β-Amylase. 

Figure 1. From Viader et al (2021) (a) α-Amylase and (b) β-Amylase activity across a series of mashing profiles, performed at lab scale. The activity was measured with the Brewer's Dream™ kit. The mash out temperature was 78 °C (1 min). 

The authors used eight temperature profiles, one of which is representative of practice in industry (no protein rest, a β-amylase rest at 65 °C & an α-amylase rest at 72 °C). The seven other profiles varied in the steps that promote β-amylase extraction during the mash and shorter β-amylase rest (at lower temperatures). Data presented (Figure 1), shows that the most succinct mashing profile yielded similar conversion rates as the original (longer) version(s). 

The shortest mashing profile results in a 20-min mash time reduction and, importantly, did not seem to affect the concentration of fermentable sugars and free amino nitrogen (FAN) levels. The authors, therefore, decided to test and compare their new mash schedule in a pilot brew experiment.

Figure 2 From Viader et al., 2021. (a) α-Amylase and (b) β-Amylase activity in mashing profiles applied at pilot scale. Measurements were taken at timepoints, equivalent to those in lab-scale experiments. The Mash heating rate was 1 °C/min for lab-scale experiments, and 2 °C/min for pilot-scale experiments. Mash out temperature was 78 °C, which was held for 1 min.

The authors confirmed their findings in a pilot-scale brew. The resulting beer was comparable to the industrial mashing program in ethanol and foam stability. The predictive value of their experimental setup, combined with ease of use, should enable brewers to (re) evaluate their mashing schedules. That's an opportunity to save on costs, right there! 

How does the kit work?

These results then beg the questions, what is the kit, and how does it work? First and before I go into this, I should stress that I do not endorse brand nor product. I haven't worked with the kit myself and have no links to those who sell or use it.

The authors used GlycoSpot Brewer's Dream kit, an analytical kit that allows you to measure the conversion of starch to simple sugars. The kit works based on a colour change that occurs when simple sugars (the breakdown products of either α-Amylase and β-Amylase) bind to indicator compounds present in the reaction mixture.

Experimental flow diagram for the GlycoSpot Brewers Dream kit (obtained here). A series of sample processing steps allows for the measurement of amylase-dependent conversion of starch into simple sugars. Spectrophotometry can then be used to measure levels of breakdown products after incubation. By comparing values from experimental samples, to those obtained from negative controls and a standard (positive controls), one can derive the level of amylase activity in your sample.

By measuring the change in colour in a given sample and comparing it to the (rate of) changes seen in positive controls (samples into which commercially produced α-Amylase or β-Amylase are added to the substrate), one can estimate the level of enzyme activity in a sample. An electronic device called a spectrophotometer-plate reader can measure the absorbance of light at specific wavelengths, 96 samples at a time. If within a linear range, you can then use absorbance values to calculate the level of breakdown products. This concentration of simple sugars, arising from degradation caused by the Amylases, acts as a proxy for enzyme activity. Whilst there are more direct methods to measure enzyme activity (using assays that measure the actual conversion rate by measuring over short time intervals), this approach is adequate for most purposes.

Anything else I should know?

The methodology is fairly straightforward if you know what you are doing. Cost associated with running the experiments can add up though. Besides the kit, some basic reagents are needed which are perfectly affordable. Unfortunately, measurements require a plate reader (fairly expensive) and a basic lab setup (pipettes, rotary shakers, centrifuge) that many breweries may not have and require investment. Anyone wishing to go down this route could benefit from a chat with someone who is knowledgeable in this arena (hint hint 😉). If done properly, you may find that there is scope to make your process greener and more cost-effective.

I hope you enjoyed reading this piece. Have a great weekend!

Cheers!

Edgar, The Beerologist.