For the first 5,000 years that humans made and consumed beer, little was known about the actual scientific principles involved in its manufacture. Beer making was an art, practiced by craftsmen. Only in the last 150 years have biochemists and microbiologists identified the relevant organisms and metabolic pathways involved in the beer fermentation. In the past ten years alone, the entire genome of Saccharomyces cerevisiae (albeit, a lab strain, not an actual brewing strain) has been sequenced, with nearly half of the genes now having an assigned function.
This sequence information is now being used to understand yeast physiology, especially as it relates to brewing (Box 9-10). However, despite this knowledge, and the thousands of years of "practice," the brewing process is still far from perfect, and producing consistent, high-quality beer is still a challenge, even for large, highly sophisticated brewers. In addition, economic pressures, quality concerns, and perhaps most importantly, new market demands, have led the industry to consider new ways to improve the beer-making process. Advances in molecular genetics and biotechnology have made it possible to address these challenges via development of new brewing strains with novel traits and tailored to perform in a specific manner.
Box 9—9- Metabolic Engineering Approaches in the Manufacture of Non-alcoholic Beer
Two general processing approaches have been used to make non-alcoholic beer. One involves removing or separating ethanol from beer by physical means (e.g., distillation, evaporation, and reverse osmosis).The other approach has been to curtail the fermentation such that little or no ethanol is produced. Both of these approaches have their pitfalls, as already described.
The availability of molecular tools has now made it possible to consider biological strategies to produce non-alcoholic beer. Although the selection of brewers' yeasts has been based, for hundreds of years, on their ability to ferment wort sugars to ethanol, recent studies have shown that it is possible to redirect metabolism away from ethanol production and toward synthesis of other end products.
During the beer fermentation, several end products, other than ethanol, are ordinarily produced. Glycerol, in particular, can reach concentrations of more than 2 g per liter. Lesser but still relevant amounts of acetaldehyde, 2,3-butanediol, and acetoin are also formed.These products are derived from the glycolytic intermediates, dihdyroxyacetone phosphate (DHAP) and glycer-aldehyde-3-phosphate (G-3-P), at the expense of ethanol (Figure 1).
Glycerol, for example, is formed from G-3-P via glyceraldehyde-3-phosphate dehydrogenase (GPD) and glycerol-3-phosphatase (GPP). By diverting even more of the glucose (or maltose) carbon to these alternative pathways, less ethanol would theoretically be produced. In the approach adopted by Nevoigt et al., 2002, the GDP1 gene encoding for GPD was cloned and over-expressed in an industrial lager yeast strain.The transformants produced more than four times the amount of GPD, compared to the parent strain, and more than five times more glycerol. Importantly, the ethanol concentration in a typical brewing wort during a simulated beer fermentation was reduced by 18%, from 37 g/L to 30 g/L.This reduction, however, was less than half that achieved previously when GPD was over-expressed in a laboratory strain of Saccha-romyces cerevisiae (Nevoigt and Stahl, 1996).
There were also large increases in the concentrations of acetaldehyde and diacetyl during the primary fermentation, and although these levels decreased during a subsequent secondary fermentation, they were still high enough to affect the flavor in a negative way.Thus, more metabolic fine-tuning of the competing pathways will be necessary to engineer a yeast capable of producing nonalcohol beers.
A completely different strategy for making non-alcoholic beer also was described by Navrâtil et al. (2002).Their approach was based on the knowledge that: (1) non-alcoholic beer is sensitive to microbial spoilage; (2) low wort pH is inhibitory to contaminating microorganisms; and (3) addition of lactic acid or lactic acid bacteria to the wort stabilizes the beer. Because acidification of wort with lactic acid or lactic acid bacteria is either not allowed or is difficult to control, another way to promote acidification was needed.
Therefore, strains of S. cerevisiae defective in enzymes of the tricarboxylic acid (TCA) pathway and known to produce elevated concentrations of organic acids were used under simulated batch or continuous fermentation conditions (and using free or immobilized cells). In all cases, the beer pH was 3.25 or less when the mutant cells were used (compared to pH 4.1 to 4.2 for the control strain).Although the mutations were not located within ethanol production genes, the test strains produced very low amounts of ethanol (<0.31% for free cells and <0.24 for immobilized cells).The latter result presumably was due to the inhibition of ethanol formation, specifically, pyruvate decarboxylase and alcohol dehydrogenase, at low pH. Although other end products, including diacetyl, were also produced, informal sensory analysis suggested that the beer compared favorably to conventionally-produced nonalcoholic beer.
Box 9—9- Metabolic Engineering Approaches in the Manufacture of Non-alcoholic Beer (Continued)
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