Mitochondrial Membrane Potential and Aging in the

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•Isolating wild yeast may lead to the discovery of a new species. •Wild yeast may ... Wort: Unlike the characteristic facial feature of witches, wort is a specialized ...
Mitochondrial  Membrane  Potential  and  Aging  in  the   Beer  Fermentation  Process 543.12/4638/B544

Email:

David  J  Hall

[email protected]

Department  of  Chemistry,  Lawrence  University,  Appleton,  Wisconsin,  54911-­0599

• Determine the effects of different parameters on mitochondrial membrane potential and aging.

METHODS Isolation

Collect  plant   and  substrate   matter

Grow  in  YPD

•Paramount  to  the  brewing  process: • Fermentation  of  sugars  to  make  alcohol  and  flavors • Intrastrain  differences  and  hybridization  are  key  in  cultural   beer  differences. • Translational  brewing:  By  studying  the  biochemical   processes  of  different  yeasts  through  a  brewing  lens,  we   can  enhance  the  brewing  process  while  gaining  information   on  their  biochemical  function.

Wild  Yeast  Isolation •Wild  yeast  can  be  found  in  countless  environments,  and  isolating   them  allows  us  to  analyze  the  biochemistry  within  genera  and   species. •Understanding  the  biochemistry  contributes  to  our  knowledge  of   the  role  different  yeasts  play  in  specific  niches  and  environments. •Isolating  wild  yeast  may  lead  to  the  discovery  of  a  new  species. •Wild  yeast  may  impart  unique  flavors  or  fermentation  profiles  in   the  brewing  process.

Plate  Samples

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Geo  Mean

Geo  Mean

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Day

YPD:  565  MMP  Zn

YPD:  565  MMP  DAP 600

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• We define a generation as 3 days (complete attenuation) and re-­pitch a sample of the yeast from the bottom as brewers do into a new pico brewery every 3rd day (75 ml of wort was used).

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Day

100

We  mimicked  the  brewing  process  for  10  different     yeasts  using  air-­locked  pico breweries:

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Wort:  End  of  Generation  WGA 40

• Measurements of MMP/WGA are taken daily from samples from the middle and bottom of the breweries.

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PCR  and   Sequence

Inoculate  into   YPD

Streak   individual   colonies

Geo  Mean

25

• Any treatment is added at the start of every generation.

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830 565

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Wort  vs.  YPD

838 833

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Mitochondrial  Membrane  Potential  (MMP)   Assays • MMP is the proton gradient formed in the intermembrane space of the mitochondria when NADH provides electrons.

Wort: Unlike the characteristic facial feature of witches, wort is a specialized growth medium for beer yeast with a variety of sugars, including maltose. The composition of wort depends on the grains used and plays an important role in the style of the beer (eg porter vs. lager) YPD: The standard media in the laboratory to culture yeast. The most important carbon source is glucose.

Aging  Assays  with  Wheat  Germ  Agglutinin

• We measure MMP using flow cytometry to evaluate the amount of green-­shift fluorescence of Rhodamine 123 which emits less fluorescence when bound to protons. • A high MMP (low geo mean) suggests a better functioning mitochondrial membrane.

• Yeast reproduce asexually by budding. • Each time a yeast reproduces, it leaves a bud scar. • Wheat Germ Agglutinin (WGA) is an protein that binds to bud scars and is labeled with a fluorophor. • Adding WGA and analyzing samples with flow cytometry reveal the fluorescence from bud scars and thus the age of the yeast.

• -­NAD+/NADH Hypothesis: more NADH= synthesis of more flavor compounds.

RESULTS

(http://www.nature.com/scitable/topicpage/mitochondria-­14053590)

Flow  Cytometry

•Isolating  wild  yeast  leads  to  a  collection  of  isolates  for  further   research.

Wild  Yeast  Contribution  to  Beer We often don ’ t realize what the wild brethren of our domestic plants and animals look like. For example, wild strawberries are no bigger than your thumb, and wolves are much larger than your average dog. Thus, it’s important to understand the genetic contributions of “ wild ” things compared to their domestic cousins. In an effort to better understand fermentation yeasts, we isolated 700 different wild yeasts from 3 continents, 7 states and 12 countries.

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Why  Yeast? •Model  organism  used  to  study  countless  biological  and   biochemical  mechanisms

Pico  Brewery

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Geo  Mean

• Evaluate the longevity of different yeasts by measuring bud scars through Wheat Germ Agglultinin (WGA) assays.

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Geo  Mean

• Biochemically evaluate the importance of mitochondrial membrane potential as a way to distinguish different yeast and monitor the brewing process

Like translational medicine the objective is to apply basic research to a problem of economic importance. The literature is replete with thousands of papers on yeast 1 2 3 4 5 6 biochemistry and yet little of that Generation knowledge has been applied to industrial scale brewing. Our aim is to evaluate mitochondrial membrane potential (MMP) and aging (WGA) as 2 measures of yeast viability and productivity during the fermentation process. MMP and WGA data were gathered from industrial sized fermenters at Stone Arch Brewery and compared to data collected in our own pico breweries.

Wild  Yeast  Isolate  Profiles ID  #

Yeast  Species

Location

Temp

Flavor

AZ7

Torulaspora  delbrueckii

N.  MI  Seyval  ground  upper

RT

Sour

JD2

Rhodotorula  fujisanensis

N.  MI  Traminette  vine

4C

Funky

BP5

Saccharomyces  cerevisiae  isolate

N.  MI  Traminette  ground

30C

Cloves

BN7

Saccharomyces  cerevisiae  isolate

N.  MI  Vignole  lower  ground

30C

Fruity

DV1

Saccharomyces  cerevisiae

Costa  Rica  fruit  stand

30C

Earthy

FE1

Rhodotorula  nothofagi

N.  MI  Vignole  vine  upper

RT

Funky

M3

Saccharomyces  cerevisiae

Pensacola,  FL  state  park

30C

Fruity

Specific  Gravity

• Cell  samples  measured  one  cell  at  a  time  (30,000  cells) • Lasers  used  to  detect  fluorescence  (background,   fluorophor– eg.  Rhodamine  123)   • Measure  of  size,  complexity,  vitality,  MMP  and  WGA

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CONCLUSIONS Specific  wild  yeast  isolates  impart  unique  flavor  and   fermentation  profiles  to  the  brewing  process  with   differing  attenuations  both  before  and  after  the  addition   of  Stone  Arch  industrial   yeast  1098. The  Status  of  MMP  in  pico  breweries  is  reflected  on  an   industrial  scale. • A  peak  in  geo  mean  (decrease  in  MMP)  is  apparent  in  both   Industrial  and  wort  pico  brewery  samples  (sometimes   multiple  peaks  in  picos). MMP  values  differ  across  generations  with  different  types   of  yeast,  different  mediums   (wort,  YPD),  and  treatments   of  DAP,  Zn,  Cu  and  Lithocholate. • Eg.  Zn,  DAP  increase  geo  mean  (decrease  MMP)  of  565  in   YPD.  MMP  of  565  in  YPD  is  more  variable  and  not  as   uniform  between  middle/bottom. Different  yeast  have  unique  aging  in  wort  pico  breweries,   with  some  similar   decreases  in  budding  as  the   generations  accumulate.  

ACKNOWLEDGEMENTS

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We  would  like  to  thank  Kali  Thibault  and  Daniel  Martin  for  their   contribution  to  this  research.  We  also  recognize  Stone  Arch   Brewery  (Appleton,  WI)  for  their  collaboration.

1.04 Specific  Gravity  (g/cm3)

BACKGROUND

• Isolate, identify and characterize wild yeast.

YPD:  565  MMP

Wort:  565  MMP  

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Geo  Mean  of  Fluorescence

The  process  of  fermentation  and  the  characteristic   aromatic  compounds  elicited  play  a  key  role  in  the  flavor  of   bread,  beer  and  wine.  However,  the  practice  of  fermentation,   particularly  as  it  applies  to  beer,  is  more  art  than  science.  The   quality  of  the  resulting  products  of  fermentation   rely  on  the   physiological  state  of  the  yeast  biomass,  that  is,  its  vitality.  A   central  organelle  to  vitality  that  is  often  overlooked  is  the   mitochondria.    Mitochondria  are  amazingly  dynamic  in  their   morphology,  distribution  and  membrane  potential  as  they  reflect   the  energy  requirements  of  the  cell.  Two  key  measures  of  yeast   vitality  are  mitochondrial  membrane  potential  and  aging.  We   hypothesized  that  there  are  differences  in  these  two  key   measures  of  vitality  based  on  the  domestication  of  the  yeast   (lager,  ale,  Belgian),  the  number  of  times  the  yeast  was  used   (generations)  and  the  type  of  supplements  used  (Zn,  Cu,   lithocolate,  diammonium phosphate).  Our  findings  provide  an   explanation  to  several  beer  brewing  observations  and,  coupled   with  bioinformatics  of  the  mitochondrial  genomes  (lager,  ale   Belgian),  provide  an  avenue  to  study  the  molecular  basis  of   yeast  vitality  as  it  applies  to  beer  fermentation.  

Translational  Brewing

OBJECTIVES

ABSTRACT

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Before  1 098 After  1 098

1.01 1 0.99 AZ7

JD2

BP5

BN7 YEAST

DV1

FE1

M3