The Science of Brewing: Malting, Mashing, and Fermentation

The Chemistry of Starch

To understand mashing, one must understand the structure of the starches involved. Starches are polysaccharides—long chains of glucose molecules linked together by glycosidic linkages. There are two main types of starch in barley:

1. Amylose: A straight-chain polysaccharide where glucose units are connected primarily by $\alpha$-1,4 glycosidic linkages.
2. Amylopectin: A branched polysaccharide. While it also uses $\alpha$-1,4 linkages for its chains, it contains $\alpha$-1,6 glycosidic linkages at the points where branches occur.

In these chains, the carbons are numbered 1 through 6. The carbon at position 1 is known as the reducing end, while the ends of the chains that do not have a free carbon 1 are called non-reducing ends. Because amylopectin has branches, every branch point creates a new non-reducing end, providing more "starting points" for certain enzymes.

The Role of Amylase Enzymes

Two specific enzymes facilitate the breakdown of these starches, and they behave differently based on the environment of the mash:

• $\alpha$-amylase: This enzyme acts randomly along the starch chains, cutting them at various points. It is most active at higher temperatures and higher pH levels.
• $\beta$-amylase: This enzyme is more precise; it clips glucose units two at a time starting from the non-reducing ends. However, $\beta$-amylase cannot bypass the branch points created by $\alpha$-1,6 linkages. It is favored by lower temperatures and lower pH levels.

Exam Tip: If a question asks which enzyme is responsible for "random" cleavage versus "systematic" cleavage from the ends, remember: $\alpha$ is random/high temp/high pH, and $\beta$ is systematic/low temp/low pH.

Fermentation: From Sugar to Alcohol

Once mashing is complete, the liquid (now called wort) is filtered to remove the spent grain. Next, yeast is added to the liquid.

Yeast performs fermentation, a biological process where the microorganisms consume the simple sugars produced during mashing. It is important to note that yeast can only ferment the smallest, simple sugars; larger, unreacted starches remain in the solution.

The chemical result of this process is the production of ethanol (alcohol) and carbon dioxide ($CO_2$). For every single molecule of glucose ($C_6H_{12}O_6$) consumed, the reaction produces two molecules of ethanol and two molecules of carbon dioxide.

We can verify this by looking at the carbon balance. Since glucose has 6 carbons:

• Two molecules of ethanol ($C_2H_5OH$) provide $2 \times 2 = 4$ carbons.
• Two molecules of carbon dioxide ($CO_2$) provide $2 \times 1 = 2$ carbons.
• Total: $4 + 2 = 6$ carbons, matching the original glucose molecule.

Analytical Concepts in the Lab

To monitor the progress of brewing, scientists use specific analytical tools to measure sugar concentrations.

Spectrophotometry and Absorbance

A spectrophotometer is used to measure the concentration of reducing sugars in a sample. This is done by measuring absorbance—how much light the solution absorbs at a specific wavelength. In these assays, absorbance is directly proportional to the concentration of the sugar; as the concentration increases, the absorbance increases.

The Dilution Factor

Often, a sample is too concentrated to be measured accurately by a spectrophotometer. In these cases, we use a dilution factor to reduce the concentration. The dilution factor is defined as the ratio of the initial volume to the final volume:

$\text{Dilution Factor} = \frac{V_{initial}}{V_{final}}$

Worked Example: If you have a sample and you want to perform a ten-fold dilution (a dilution factor of 1/10) to reach a final volume of $200\ \mu\text{L}$, how much starting solution do you need?

Using the relationship: $V_{initial} = V_{final} \times \text{Dilution Factor}$ $V_{initial} = 200\ \mu\text{L} \times \frac{1}{10} = 20\ \mu\text{L}$

To complete the solution, you would add $180\ \mu\text{L}$ of water to your $20\ \mu\text{L}$ of starting solution to reach the final $200\ \mu\text{L}$ volume.