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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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).
7
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.
300
Day
Why Yeast? •Model organism used to study countless biological and biochemical mechanisms
Pico Brewery
350
Geo Mean
• Evaluate the longevity of different yeasts by measuring bud scars through Wheat Germ Agglultinin (WGA) assays.
350
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
1.05
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
1.03 1.02
Before 1 098 After 1 098
1.01 1 0.99 AZ7
JD2
BP5
BN7 YEAST
DV1
FE1
M3