The Biotechnology of Brewing

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The Biotechnology of Brewing

• Matthew Rowe - PhD Candidate
• Contact ph x7887
• or email m.rowe_at_auckland.ac.nz

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Lecture Summary

• Lecture 1 - Introduction and History
• Lecture 2 - Overview of Brewing process
• Lecture 3 - Brewing Processes Continued
• Lecture 4 - New Developments within the Brewing
  Industry

Major References
1. Priest, F.G. and I. Campbell, eds. Brewing
microbiology. 2nd ed. . 1996, Chapman Hall
Falmouth, Cornwall. 2. Hough, J.S., The
biotechnology of malting and brewing. 1991,
Cambridge University Press. 3. Doran, P.M.,
Bioprocess engineering principles. 1st ed. 1995,
London Academic Press Ltd. 4. http//LionNathan.
co.nz
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Introduction

• Brewing is a traditional biotechnology
• Incorporates a number of disciplines

Chemistry
Biology
Brewing Biotechnology
Marketing
Engineering
Management
An industry with it roots firmly embedded in
history Well over 10 000 000 000 litres produced
world-wide annually
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Brewing pre 1900s

• Brewing process discovered over 5-8000 years ago
• Earliest recorded evidence of brewing is by the
  Egyptian / Babylonian civilisations (2300 BC)
  (Figure 1)
• Recently breweries have recreated beer formulated
  from the sediment found inside an ancient brewery
  inside the Sun temple of Nefertiti, queen of
  Pharoah Akhenaten by two scientists and an
  Egyptologist working for Scottish Newcastle.
• Although brewing is a complex process its
  technology does not need to be

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• Domestication of grain during early Neolithic
  period a result of bread or beer production?
• In 1516 the Reinheitsgebot was designed to
  protect the consumer from beers brewed with
  low-quality ingredients (called adjuncts) such as
  rice and corn
• In actuality, there are only three ingredients
  mentioned in the original text barley, hops, and
  water.
• In 1516 the brewer's knowledge didn't extend to
  microbiology, so since they didn't say anything
  about yeast, there are no truly Reinheitsgebot
  beers today, technically speaking!
• Germany the only country still adhering to these
  laws. The use of adjuncts is widely accepted in
  most modern breweries
• Fermentation was not understood until 1857, when
  Pasteur discovered it was caused by a living
  microorganism, yeast.
• Disproves spontaneous generation
• In 1897 Eduard Buchner successfully prepared a
  cell-free yeast extract capable of fermentation
• i.e. discovery of enzymes - birth of modern
  biochemistry
• In 1883 Emil Hansen introduces pure culture
  propagation equipment

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Types of Beer

• Two major types - separated by fermentation
  characteristics
• Lagers( to lay down or store)
• Ales
• both can be either light or dark in appearance
• Lager
• Produced with Saccharomyces uvarum
• Yeast flocculates to the bottom - recovered by
  cropping
• Yeast generally discarded after 8-12 generations
• Pure culture fermentations
• Fermentation temperature generally cold - 8-120C
• Generally produces a superior product
• Most beer produced is of the lager variety

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• Ale
• Produced with Saccharomyces cerevisiae
• Yeast flocculates to the top - recovered by
  skimming
• Yeast reused after each batch
• Mixed culture fermentations
• Fermentation temperature generally warm (16-240C)
• Produced mainly in Great Britain and Continental
  Europe
• A huge variety of beers exist. Changes in any of
  the process conditions can result in vastly
  different beer styles
• Beers can be classified by region and original
  gravity (OG)
• OG refers to the amount of sugar present prior to
  pitching (yeast inoculation)
• OG determines final beer alcohol content
• 1.008 Specific gravity (SG) 2 sucrose 20
  Plato

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Lion Beer Spectrum
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Yeast

• Single most important ingredient - no yeast no
  beer!
• Historically the unknown ingredient (x)
  inoculation occurred by accident or by
  reintroducing skimmings
• Not mentioned in the Reinheitsgebot.
• Yeast Definition
• Generally accepted as uni-cellular fungi
• Complex classification historically based on
  physiology and morphology characteristics.
• Saccharomyces cerevisiae species now consists of
  many hundreds of different strains
• Modern classification and species differentiation
  achieved by DNA fingerprinting
• Species differentiation very important for the
  brewery

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• Yeast Growth Requirements
• Sufficient nutrients and growth factors
• Moderate temperatures
• Moderate pH
• Absence of inhibitors / toxins
• Osmotic pressure not excessive
• Growth of yeast is through cell division -
  budding - once cell size reaches critical value
• Cytoplasm of the new cell bulges out through a
  weak area of the mother cell
• Daughter cell is formed through migration of
  duplicated cellular organelles
• Bud once fully grown detaches leaving a bud
  scar
• Growth both increase in cell mass number
• Flocculation of the yeast
• Due to chains of cells not detaching after
  budding
• Top-fermenting yeast carried up by CO2
• Flocculation enhanced by bringing calcium or
  carbonxyl groups between cells (hydrogen bonding)

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• Yeast Growth is dived into five distinct phases
• Yeast Maintenance within the Brewery
• Pure yeast generally kept on site as stock
  cultures stored under liquid N2, on agar slopes,
  or lyophilised.
• These pure yeast stock cultures used to replenish
  the brewery with fresh yeast
• Yeast cropped from fermentations or propagation's
  stored at low temperatures in agitated holding
  tanks prior to pitching

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• Propagation of Pure Yeast Cultures
• Typical batch propagation procedure is as follows
• Why Repropagate Yeasts
• Mutation
• Microbial infection / contamination
• Loss in viability - High DCC
• Loss in vitality
• Atypical beer production
• There are however many limitations to this
  procedure which will be discussed later along
  with new technologies for propagating brewers
  yeasts

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• Nutrient Uptake
• As mentioned the cell wall separates the yeast
  from the environment
• Some compounds / molecules pass freely across the
  cellular membrane i.e proteins, hop residues,
  polyphenols, ethanol.
• Others require transport through either active or
  passive protein mediated complexes.
• Generally the more readily assimilated compounds
  are used / transported first. for example amino
  acid utilisation will occur in a sequence, the
  same applies for sugar utilisation.
• Yeasts also require a variety of trace elements
  for example zinc, magnesium, calcium, --gt enzymes
• Yeast Metabolism (in brief!)
• Gay-Lussac Equation
• Glucose ? 2 CO2 2 EtOH Energy
• More realistic expression (g)
• Maltose(100) Amino Acid (0.5) ?
• Yeast (5) Ethanol (48.8) CO2 (46.2) 50kCal
• Glucose dissimilation (glycolysis) occurs through
  the Embden -Meyerhof-Parnas (EMP) pathway
• Under anaerobic conditions pyruvate metabolism is
  directed into acetaldehyde
• Under aerobic conditions terminal electron
  accepter is available and metabolism is directed
  through the Krebs cycle and onto the mitochondria

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• Not all glucose is metabolised by the EMP pathway
  - Hexose monophosphate pathway or pentose
  phosphate pathway- important for the production
  of pentose sugars (nucleotides)
• Production of higher alcohols, esters and acids
  occurs through reaction of intermediates of these
  pathways.
• These have important effects on the final flavour
  of the finished product
• One of the most important is diacetyl
• Acetolactate is excreted into the wort during
  fermentation - decarboxylates (chemically) into
  diacetyl (CH3COCOCH3)
• During maturation of beer this compound diffuses
  back into the cell by a reductase enzyme NADH2
  to produce 2,3 butanediol
• Considerations during Fermentation of Wort
• CO2 recovery important, avoids potential hazards
  (4), and enables recycling for packaging
• Cooling essential - 3.5 kJ L-1 h-1
• Healthy yeast pitched quickly on to avoid growth
  of contaminating microbes (spoilage)
• High pitching rates to shorten lag phase

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• The nature of a good brewing yeast
• rapid fermentation rate without excessive growth
• efficient utilisation of maltose and maltotriose
  with a good yield of ethanol
• ability to withstand ethanol and osmotic shocks
• reproducible product - flavour and aroma
  (consistent)
• ideal flocculation character for the process
  employed
• Good handling characteristics
• Yeast Improvement
• Altering yeast biochemistry (diacetyl)
• Altering yeast physiology (high gravity,
  immobilised cell bioreactors)
• Development of new strains
• Above can be achieved through
• Mutation and Selection
• Hybridisation
• Rare Mating
• Spheroblast fusion
• Transformation, recombinant DNA

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Barley

• Why Barley?
• Other grains tend to either produce rancid
  material or are subjected to heightened microbial
  contamination
• Flavour of malted barley appreciated over other
  grains
• High starch content high extract (sugar)
• High protein content
• Germination of barley releases malt enzymes
• ? and ? amylases
• responsible for conversion of large starch
  molecules into low molecular weight sugars
• Provides all the necessary nutrients for yeast
  growth

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Hops

• Humulus lupulus a member of the Cannabinaceae
  family. However it contains no hallucinogenic
  substances.
• The cones of female plants are harvested
• The lupulin glands contain the bitter resins
  (humulones or ?-acids) and essential oils.
• Hops are added to the brewing cycle during wort
  boiling in order to extract their bitter
  compounds
• Good quality hops contain
• high concentrations of ?-acids (kettle hops)
• attractive aroma (aroma hops)
• Both high ?-acids and aroma (mixed hops)
• ?-acids are the most important source of
  bitterness in beer
• during boiling the ?-acids are rearranged
  chemically to form iso ?-acids (iso humulones)
  which are substantially more bitter

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• ?-acids (lupulones) not as important
• Essential oils comprise of hundereds of compounds
  which generally evaporate during wort boiling
• include terpenes, myrene, farnesene, and humulene
• hops are only 1 essential oil but this is almost
  100 of their aroma
• Although hops can be added in their natural state
  to the kettle, hop processing is very common and
  hop pellets and extracts are preferred by many
  large breweries, their advantages include
• good storage
• very little oxidative degradation
• easily blended with other varieties
• easily weighed and conveyed

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Water

• Makes up 95 of beer
• Also used extensively throughout the brewing
  process
• Cooling and heating water
• Cleaning
• Some 4-10 times the amount of water is used per
  volume of beer produced
• Financial incentives to conserve water
• Historically the breweries would be placed close
  to a reliable water supply
• Thus different styles of beer developed due to
  the mineral content of the water i.e. high
  calcium sulphate content of Burton-on-Trent
  produced full-flavoured strong pale ale
• Advent of chemical analysis of water enabled any
  brewery to mimic water of others

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• Calcium and carbonate concentrations in water
  important for wort pH
• Calcium stabilises malt ?-amylase, without Ca2
  it fails to function efficiently
• Calcium also aids wort pH reduction though
  precipitation of calcium phosphate (phytin
  ?phosphoric acid which disassociates, H
  released, pH falls)
• Water contamination
• Microbial and chemical contamination of water
• Chemical run-off from farms into brewery
  catchment areas
• Close monitoring of water quality into the
  brewery can alleviate potential fermentation
  problems downstream
• Microbes generally eliminated through filtration
  or chlorination

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Adjuncts

• Adjuncts are defined as additions to the wort
  other than barley, hops, yeast or water
• Used for several reasons
• cheaper sources of extract than malt, increased
  OG
• If nitrogenous material present in excess in
  original malt adjuncts serve to dilute them to
  appropriate -levels (excess nitrogen material --gt
  haze)
• Improved flavour stability
• Beers more refreshing (impt for lagers)
• Improve or change the final beer colour
• Improved beer foam
• Both solid and liquid adjuncts are commonly used
• Solid Adjuncts
• Most common derived from maize or rice
• Added to the mash-tun
• Generally solid adjuncts require separate intake,
  storage and milling facilities thus considerable
  cost savings can be made by utilising liquid
  adjuncts

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• Liquid Adjuncts
• Maize processed to produce glucose syrups or
  the products of starch hydrolysis
• Starch slurry is liquefied by adding bacterial
  ?-amylase and applying heat (850C)
• This results in a solution with a range of sugar
  molecules from glucose to dextrins
• Addition of other industrially grown enzymes
  further breaks down the hydrolate
• Cane and beet sugars also widely used (pure
  sucrose or fructose / glucose (acid hydrolysed
  sucrose))
• Syrups generally stored at high temperatures to
  help pumping
• Syrups added to the kettle during or after wort
  boiling and prior to the wort cooling (heat
  exchangers) to help with dissolving
• High gravity brewing
• Addition of adjuncts increases OG of wort
• Heating and cooling higher gravity wort requires
  very similar energy expenditure compared to LG
• Producing beer with HG wort results in stronger
  alcohol beer which can be diluted later - reduces
  production vessels size
• Two methods
• Add syrups to wort kettle
• Or divert LG wort (lt1.010) back into mash-tun in
  place of water

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• Problems with HG Brewing
• Losses of worts / beers due to poor collection
  etc
• Hops bitters under utilised in HG worts
• Yeasts produce different flavours
• When blending beer and water it is hard to
  achieve a consistent product over time