Friday, May 17, 2013

Biology of Beer

There's an ancient battle raging everyday in breweries around the world.  The battle is to protect your beer from dastardly bacteria that want to invade and spoil it.  We're not fighting alone, though.  The yeast that ferment the beer do a lot of the antibacterial work themselves.  They make beer into a pretty inhospitable environment for bacterial growth, with a low pH and lots of alcohol, but bacteria are tough little buggers.  Let's take a little journey through some of the science of beer.  This post is a salute to the complex microbiology of beer and beer brewing as well as the men and women who developed and continue to develop modern brewing.  Open a cold one and read more after the jump....         

The magic and mystery of yeast

No one knows exactly how beer originated, but it is thought to be the oldest fermented beverage.  It is very possible that grain stocks contaminated with natural yeasts just happened to ferment, perhaps after getting wet during a flood.  Eventually, some adventurous soul tasted the resulting liquid and decided it was good, maybe even great, but it is hard to believe that he or she could have realized that this discovery would change the world.  The brewing of beer has been called mankind's oldest form of biotechnology, though for most of history we didn't understand anything about the biology behind the technology.  We know that the fermenting of various grains, which I'll generally call "brewing," goes back at least thousands of years.  Archaeologists have discovered ancient Sumarian tablets from 3,000 years ago, perhaps even older, that contain a hymn to the ancient Sumerian goddess of brewing, the "Hymn to Ninkasi."  Ancient Romans, Babylonians, and Egyptians are also thought to have also made beer-style beverages from barley and other grains.

While the brewing started thousands of years ago, ancient people certainly didn't understand what was going on or how their beer was really made.  They often attributed the formation of beer to magic or divine intervention.   To them it was a miracle.  They knew that the process produced a magical ingredient that made them feel good (alcohol) and also a fizzy gas (carbon dioxide).  In fact, the gas release during this process is what gave us the word ferment, which comes from the Latin fervere,  which means "to boil."     
We now know that the critical agent in beer fermentation is yeast.  Yeasts are unicellular (single-celled) fungi, usually less than 5 microns in diameter (a micron is one millionth of a meter), far too small to see with the naked eye.  Even though yeast are unicellular, they are not the same as bacteria.  Yeast cells are much more like human cells than bacteria are.  In fact, many biology researchers use yeast cells to study basic cellular processes that also occur in human cells.   

Yeast can make energy by fermenting sugar to make alcohol.
Even though many people think of yeast coming from the grocery store in little packets, yeast live in the wild, where they can usually be found near a sugary-food source like the skin of berries or fruits.  Yeast reproduce by budding, a process where a smaller yeast cell (called the "daughter cell") buds off of the "mother cell" to create a new yeast cell.  This requires energy.  Yeast obtain the energy to grow and reproduce by converting the simple and complex sugars found in fruit, grains, or likewise beer into alcohol and carbon dioxide through the process of fermentation.  The energy that they get from fermentation comes from making molecules called adenosine trisphosphate, also known as ATP.  This ATP the same molecule that all cells, even human cells, use for energy.  Carbon dioxide and ethyl alcohol, also known as ethanol or simply alcohol, are byproducts of this process.  As important as these two molecules are to us, they are merely waste products to the yeast.

At the start of the brewing process, the grains are heated in water to make a liquid called "wort," which becomes the nutrient source, or "growth medium" for the yeast.  Like other cells, yeast need a variety of carbohydrates (sugars and starches), amino acids (the building blocks of proteins), vitamins, fats and lipids, and other nutrients to thrive.  However, once they start to grow and divide, the alcohol concentration increases and the nutrient concentration decreases; the yeast growth subsequently grounds to a halt.  The resulting liquid that is left is beer, which is basically the used growth medium of the yeast.       
Through trial and error, ancient "food scientists" experimented with fermenting different kinds of grains and fruits.  They realized that if they put them in covered containers and waited, then sometimes magical things would happen.  Sometimes they would get a liquid that tasted and made them feel good.  Sometimes they would get a spoiled rotting mess.  At first the yeast for the fermentation came from whatever species were living on or around the grains.  However, there's also a lot of bacteria living in the wild.  These bacteria also want to eat the sugars in fruits and grains to get energy to multiply.  A major difference, though, is that bacteria often produce bad-tasting chemicals like lactic acid.  Ancient brewers had to experiment with the conditions like time and temperature to find conditions that worked well.  What they were doing is unknowingly experimenting to find conditions that favor yeast growth over bacteria growth.  Even though they didn't know the biochemistry behind fermentation, ancient brewers also began selecting for certain types of yeasts by using previous batches of fermentation to start new batches of fermentation.  They began the process of "domesticating" yeast.

Even with all of the brewing technology that we have, with modern breweries all over the world producing thousands of brands of beer from numerous different grains in many flavors and styles, one thing still remains necessary for beer brewing today: yeast.  Without the yeast, you don't get beer.    

Science and art combine

The history of yeast research reads like a "who's who" of early microbiology.  It should be no surprise that the importance of yeast to wine and beer resulted in a strong motivation for scientists to understand them.  Antonie van Leeuwenhoek, the "Father of Microbiology" and one of the pioneers of the microscope, first described seeing yeast in 1680 when he looked at fermenting liquid.  However, he didn't really know they were yeast at the time.  In fact, he didn't even realize they were alive.  He thought they were just bits of grain starch.  It wasn't until 1838 that Charles Cagniard de la Tour made the link between yeast and alcoholic fermentation, helped in large part by improvements in microscope technology and lens design.  He could watch the yeast multiply during fermentation, and rightly concluded that they were alive.

By 1860, Luis Pasteur, the same scientist who figured out how to preserve milk by pasteurization, figured out that alcohol and carbon dioxide are the byproducts of the yeast consuming sugar to live and multiply.  He recognized that fermentation happens when yeast consumer sugar with limited available oxygen.  Pasteur's work pioneered our modern understanding of fermentation and the role of yeast, and in 1876, he published a landmark paper titled "Studies on Beer" (E ́tudes su La Bie ́re).  Pasteur's impact on biology is legendary, and he fueled his own legacy with his extreme seriousness and obsession with his research.
From Pasteur's work and other scientific research, scientists today know that all cells, including yeast, can break down glucose to make ATP for energy.  Glucose is the same sugar that is found in your blood that your body cells also use for energy.  Many other sugars, including table sugar (sucrose) can be broken down to glucose by enzymes, which are basically just proteins that can catalyze chemical reactions and make them go faster.  Glucose is broken down by a multi-step, multi-enzyme process called glycolysis, which yields 2 ATP molecules (for energy) and 2 pyruvate (or pyruvic acid) molecules.  If there's enough oxygen around, the pyruvate can go through a process called aerobic respiration (also known as the Krebs cycle or TCA cycle) and be broken down to make more ATPs.  But, if there's no oxygen around, aerobic respiration doesn't work.  Glycolysis is the best you can do to make ATP.  However, you can see in the diagram below that breaking down glucose to pyruvate requires a molecule called NAD+ (nicotinimide adenine dinucleotide; in red), which is changed (reduced) to NADH in the process. 

Fermentation is part of an alternative respiration pathway that yeast use when oxygen is limited.

Because there's only so much NAD+ in a given cell, the cell needs to recycle the NADH back to NAD+ to be able to use it to break down more glucose to make more ATPs for energy.  Some cells can do this by converting pyruvate to acetaldehyde, which releases carbon dioxide.  Acetaldehyde can then be converted to ethanol, with NADH oxidized back to NAD+.  This is ethanol fermentation, the kind of fermentation that we want when producing beer, wine, or other alcoholic beverages.      

Ethanol Fermentation in Yeast Cells

Louis Pasteur spent a lot of time looking in the microscope at yeast cells while studying fermentation.  Because Pasteur was working many years after Charles Cagniard de la Tour,  the microscope lenses Pasteur had were much better.  Pasteur could make out different, even smaller cells in batches of fermentations that had gone bad or "sour."  In the good batches, he saw only yeast.  In the bad batches, he almost always saw the smaller cells alongside them.  This is a great example of how better technology often drives scientific discoveries, which is still true today.  He never could have discovered this had the microscope technology not been as good as it was at the time.             

The smaller cells that Pasteur saw were bacteria.  His research went on to show that there are two types of fermentations.  One type type produces ethanol (alcohol), as seen with yeast.  Another type produces lactic acid, as seen with many bacteria.  In lactic acid fermentation, pyruvate is converted to lactic acid instead of acetaldehyde.

Lactic Acid Fermentation

Lactic acid from fermenting bacteria contributes to the bad sour taste of spoiled milk.  Likewise, when bacteria ferment and produce too much lactic acid in beer or wine, it spoils and tastes terrible.

However, lactic acid fermentation isn't always bad.  This is the reaction that occurs when we mix bacteria and milk to produce yogurt, and this is also what happens when we take bacteria and cabbage to produce sauerkraut.  Lactic acid fermentation also occurs in our muscles when they need to work hard and our bodies can't get enough oxygen to the muscles quickly enough.  Muscle cells break down glucose and form lactic acid through fermentation.  Build-up of this lactic acid can make muscles feel achy and tired until our bodies can get rid of the lactic acid.  Lactic acid fermentation has its place in the world, but Pasteur showed us that a key goal of brewing is to encourage ethanol fermentation by yeast and discourage lactic acid fermentation by bacteria.  A large part of brewing research today focuses on protecting yeast cultures and beer batches from bacterial "infection."        

By the end of the 1800s, microbiology and brewing technology had both progressed to the point that brewers were using microbiological culture methods to isolate and grow pure strains of yeast to select for those that made the best beer.  Using pure strains of yeast also helped to prevent introduction of bacteria into the fermentation reaction from contaminated yeast mixtures.  The defining moment in the culture of brewing yeast came in the 1880s, when Emil Christian Hansen isolated yeasts of two different varieties, "top-fermenting" and "bottom-fermenting."  Hansen worked at the Carlsberg Laboratory in Cohenhagen, where he was able to grow pure cultures of these yeasts.  He demonstrated that these two types of yeasts yielded reproducibly different types of fermentations, which transformed the brewing industry and immensely improved batch-to-batch consistency.  In 1883, the Old Carlsberg Brewery began producing beer from isolated yeast strains, and the American companies Pabst, Schlitz, and Anheuser-Busch began to do the same by 1892.  Even today, the two types of yeasts that Hansen characterized form the basis of two of the most common kinds of beers: ales and lagers.

The top-fermenting yeast are called ale yeast, which are usually strains of the species Saccharomyces cerevisiae (abbreviated S. cerevisiae).  S. cerevisiae also is the strain used to make bread and weiss (wheat) beers.  The bottom-fermenting yeast are strains of Saccharomyces pastorianus, also known as S. carlsbergensis, which make lager.  At one time, bottom-fermenting yeast were only used in the Bavarian regions of Germany, but as German beer brewers migrated to the US, the use of these yeasts spread to other parts of Europe and to the US.  Top-fermenting yeast float during fermentation, partly due to the carbon dioxide they produce, and they form a "head" on the fermenting beer.  Bottom-fermenting yeast cells have a greater tendency to stick together or aggregate.  This aggregating and settling of yeast is called flocculation.  Yeast cells with higher flocculation will settle out of the beer faster.  The difference between these types of yeast partly lies in variations in yeast cell wall composition and the differential activity of a family of genes called FLO genes, which are also regulated by nutrient composition, pH, and temperature of the wort medium.  Yeast flocculation is complicated, and remains an active area of food research. 

Beer preservation and hops

Yeast have evolved to be surprisingly resilient.  Producing alcohol is not only a necessary by-product of fermentation but is also a natural bacterial defense mechanism.  Beer usually ranges between 4-6% alcohol, and in some cases when alcohol-resistant yeast strains are used, it can get up to 15% or higher.  This is enough to significantly impair bacterial survival.  Beer is also very acidic, with a low pH of around 3.5-4.5, also helping to slow bacterial growth.  Bacterial growth inhibition also comes from the low oxygen content and high carbon dioxide content of beer, as well as the continual decrease in nutrient content as yeast grow, multiply, and consume the nutrients in the fermenting beer.  All in all, yeast do a pretty good job of defending themselves, but bacteria are also resilient and  multiply fast.  Keeping bacteria from spoiling beer has been an ongoing battle for centuries.
Hops are a popular addition to many modern craft brews, and historically have been used not only to add bitterness and "hoppy" flavors and aromas but also increase beer's natural resistance to bacterial spoilage.  German monks used hops to flavor their beer as early as the 1100s.  Eventually people started to notice that beer brewed with hops spoiled less frequently.  Hundreds of years later, in 1516, the Lord of Bayern, Wilhelm IV, enacted a law called the "Reinheitsgebot," or the "Purity Law," today often called the "Bavarian Purity Law."  This law was meant to set standards of beer production to regulate purity and safety for consumption.  It required that beer be made from barley, water, and hops.  Historians believe that the mention of hops in the Purity Law meant to stop the use of other more shady beer preservatives, including soot and various other herbs of questionable safety.

While hops had been used as a beer preservative for hundreds of years, the antibacterial activity of hops was not scientifically demonstrated until 1888.  In 1937, a scientist named J.L. Shimwell showed that hop extracts inhibit the growth of certain types of bacteria more than others.  The hop vine produces cones that, when mature, contain granules called lupulin.  Lupulin are the most important components for the flavor and antibacterial properties of hops.  The lupulin contain chemicals called α-acids and β-acids.  The α-acids are toxic to certain types of bacteria but don't dissolve very well in water.  When the hops are boiled with the rest of the grains for brewing, the α-acids convert to iso-α-acids, which more easily dissolve into the beer.  Iso-α-acids can inhibit the growth of certain types of lactic-acid producing bacteria, called "gram-positive" bacteria because of their structure.  Two groups of gram-positive lactic acid bacteria, Pediococcus and Lactobacillus, are responsible for the majority of beer spoilage today.  The iso-α-acids of hops act as ionophores, which means they poke holes in the bacteria.  This prevents the bacteria from taking up nutrients and making energy to grow and reproduce.  However, some strains of these bacteria can develop resistance to iso-α-acids, just like some strains of bacteria found in human infections can develop resistance to certain antibiotics.  In the same way that antibiotic-resistant bacteria are a growing concern for doctors, hops-resistant bacteria are a growing concern for brewers.  Additionally, other types of gram-negative bacteria exist which are naturally resistant to iso-α-acids.  Two important groups of these bacteria that are of a concern to brewers are Pectinatus and Megasphaera, which are good at growing in low-oxygen environments such as a beer fermentation vat.

The future of beer

Research on how bacteria become resistant to iso-α-acids and other antibacterials found in hops and other beer ingredients is an active area of food science research.  Scientists want to prevent the emergence of resistant strains and/or tackle them when they do emerge.  Bacteria are relentless.  Their sole goal is to survive and multiply by any means possible, even in your beer.  However, as we use science to better understand the world around us, we are continually learning how better to deal with bacteria, including how to better keep them out of your beer. 

Brewing is a combination of art and science, and the scientists I mentioned also made significant contributions to our understanding of wine fermentation, but wine development is too large of a topic to talk about in this post.  Nonetheless, the stories of the science behind our understanding of fermentation of beer and wine are examples of how scientific research impacts many aspects of our lives, from the beverages we drink to the economic activity of the beer and wine industry.  But, as in all areas of science, even with something we take for granted like beer, there's still a lot more to learn and a lot more work to do. What we understand about the world around us is still far less than what we don't yet understand. 

© 2013 TheMadScienceBlog
Sources and further reading:
  • J.A. Barnett.  "A History of Research on Yeast 1: Work by Chemists and Biologists, 1789-1850."  Yeast.  14:1439-1451.  1998.  Available here.
  • J.A. Barnett.  "A History of Research on Yeast 2: Louis Pasteur and His Contemporaries, 1850-1880."  Yeast.  16:755-771.  2000.  Available here.
  • J.A. Barnett.  "A History of Research on Yeast 3: Emil Fischer, Eduard Buchner, and their Contemporaries, 1880-1900."  Yeast.  18:363-388.  2001.   Available here.
  • J. Kleyn and J. Hough.  "The Microbiology of Brewing."  Annual Reviews in Microbiology. 25:583-608. 1971.  (subscription or pay-per-view only)
  • E.J. Lodolo, J.L.F. Kock, B.C. Axcell, and M. Brooks.  "The Yeast Saccharomyces cerevisiae--the Main Character in Beer Brewing."  FEMS Yeast Res. 8:1018–1036. 2008.  Available here.
  • P.E. McGovern.  Uncorking the Past: The Quest for Wine, Beer, and Other Alcoholic Beverages.  University of California Press.  2010. 
  • K. Sakamotoa and W.N. Konings.  "Beer spoilage bacteria and hop resistance."  International Journal of Food Microbiology.  89:105-124.  2003.  (subscription or pay-per-view only)
  • G.C. Stewart, C.R. Murray, C.J. Panchal, I. Russell, and A.M. Sills.  "The Selection and Modification of Brewer's Yeast Strains."  Food Microbiology.  1:289-302.  1984. 

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