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Difficult to Ferment Juices

Vidhya Ramakrishnan

July 15, 2016

July 15, 2016

Factors impacting fermentations

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Difficult to Ferment juices

Problems arise even after maintaining good

practices

Routine testing and nutrient supplementation of

the must does not fix the issue

Chronic problem

Factors in difficult fermentations

Problematic Fermentations

Oxidative stress

Effect of nutritional imbalance

Influence of microbial load in the 

must

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Pathways impacted in yeast from sluggish fermentations as compared to

normal fermentations

Amino acid metabolism Biosynthesis of unsaturated fatty acids Butanoate metabolism Mannitol  Steroid metabolism Sugar metabolism TCA cycle

Mannitol

Sugar alcohol produced by plants and microbes. 

Functions Carbohydrate storage depot Osmoprotectant Scavenger of reactive oxygen species (ROS)

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Effect of Oxidative Stress on Fermentation

H2O2H2O2 H2O2H2O2

Intracellular Mannitol Accumulation

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Effect of Juice on Mannitol levels in Response to Oxidative Stress

Juice Parameters

Juice varietal Brix YAN (mg/L) YAN:YNAN ratio Pro:Arg ratio pH

Chardonnay 2013 24.9 225 0.79 12.43 3.23

Chardonnay 2015 23.3 228 1.69 5.63 3.89

Viognier 2015 29.9 330 1.31 2.48 4.12

Vermentino 2015 27.3 235 1.79 3.92 3.89

Torrontes 2015 22.1 143 1.58 2.75 3.81

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Exometabolomic analysis by GC-MS

ControlJuice with 10mM Hydrogen peroxide

Chardonnay 2015 Lower level

Viognier 2015 Unchanged

Vermentino 2015 Unchanged

Torrontes 2015 Unchanged

Gluconic acid lactone

Effect of Nutritional Imbalance on Fermentations

High proline

No/Low/high Biotin

Low Vitamins

Low Amino Acids

Total Nitrogen levels

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Summary of Effect of Combinatorial Nutritional Imbalance on Fermentations

N = Normal, S = Sluggish

Biotin Deficiency and Nitrogen Levels

Excess NH4+ can be detrimental at no/low biotin concentrations

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Impact of Bacteria in Must

Study the effect of bacteria isolated from

problematic fermentations on yeast

Assess the ability of bacterial spent media to

alter the yeast metabolic state [GAR+]

Study the effect of acetic acid and lactic acid

produced by these bacteria on yeast metabolic

state

Altered metabolic state- [GAR+] prion

Glucose repression: Genes required for utilization of other carbon sources

are switched off in the presence of glucose, Saccharomyces’ preferred

carbon source.

Wild type[gar-]

[GAR+] prion

GGM media

YeastDecreased

fermentation rate

Pma1 protein

Yeast

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Bacteria from problematic fermentations

Classify into 3 groups: • Inducing • Inhibitory • No effect

Both acetate and lactate levels produced vary across different

genera and within the same species.

UCD 175 (Acetobacter pasteurianus) shows [GAR+] inducing

phenotype in spent media assay.

1) Spent GGM media

2) GGM media

Induction of [GAR+] prion state by acetic acid

GGM plateAfter Exposure to

No acetic acid

2% acetic acid

4% acetic acid

Different levels of acetic acid exposure elicit different metabolic response by yeast

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Fermentation profiles of UCD 932 g- and [GAR+]

Sampling times

Amino acid utilization profiles

Aspartic acid (Alanine, Asparagine, Glutamic acid, Glutamine, Histidine,Isoleucine, Leucine, Phenylalanine, Serine, Threonine, Tyrosine, Valine)

Co

nce

ntr

atio

n (

mg

/L)

Day0 Day1 Day2 Day3 Day4 Day6 Day10

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Hydroxyproline (Tryptophan)C

on

cen

trat

ion

(m

g/L

)

Day0 Day1 Day2 Day3 Day4 Day6 Day10

Glycine (GABA)

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Metabolic summary of [GAR+] and g-

Amino acid utilization is slower in yeast in 

[GAR+] prion state as compared to g‐

Oxygen depletion is slower in fermentations 

with yeast in the [GAR+] prion state. 

Advantageous to bacteria! 

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Conclusions

Oxidative stress results in mannitol accumulation in yeast. The 

ability to quench the oxidative stress is juice dependent. 

Nitrogen supplementation using DAP should be done carefully. 

Effective and sufficient use of SO2 in the initial juice is 

important to alleviate bacterial impacts on yeast fermentations. 

Multiple factors in conjunction have a synergistic effect on 

yeast leading to problematic fermentation.

Acknowledgements

V&E winery staff, UCD Metabolomics Core Facility

American Vineyard Foundation (AVF)

Bisson lab