critical factors of difficult-to-ferment juices and
TRANSCRIPT
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Critical Factors of Difficult-to-Ferment Juices and
Arrested Fermentations
Vidhya RamakrishnanFEBRUARY 15, 2018
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Factors Impacting Fermentations
Yeast Metabolism
Abiotic Temperature
pH
NutritionalMacronutrients‐ Nitrogen, phosphate
Micronutrients‐ Vitamins, minerals
Microbial
Competition for nutrients
Toxic compound production
Alteration of yeast metabolism
Stress
Osmotic, High Ethanol,
Oxidative
Difficult to Ferment juices
Problems arise even after maintaining good winemaking practices
Routine testing and nutrient supplementation of the must does not
fix the issue
Chronic problem
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Factors in Difficult Fermentations
Problematic Fermentations
Stress
Nutritional Imbalance
Influence of Microbial Load in the Must
Juice Composition Typical YAN levels in normal juices‐ >120 mg/L
DTF juices have low YAN levels: < 80 mg/L
Typical Proline:Arginine ratio For non‐proline accumulating varieties‐ <2For proline accumulating varieties‐ <10
DTF juices have a very high Pro:Arg ratios: >10, even as high as 80
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Juice Amendment Results
No significant improvement in fermentation with nitrogen
amendment
This fermentation defect seen across multiple yeast strains
DTF juices
Metabolomic comparison of the
yeast from normal and DTF juices
Nutritional composition analysis
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Ionic Levels in Juices
Ionic levels in juices measured by Atomic Absorption Spectroscopy‐ Calcium ions‐ Magnesium ions‐ Manganese ions‐ Potassium ions‐ Sodium ions‐ Zinc ions
By Chloridometer‐ Chloride ions
PC 2 (41%)
PC 1 (24%)
Ionic profiles varied by region
and juice varietal
No obvious pattern observed
differentiating easy and
difficult to ferment juices
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Effect of Nutritional Imbalance on Fermentations
High proline
No/Low/high Biotin
Low Vitamins
Low Amino Acids
Total Nitrogen levels
Nutrient Study Results
Biotin deficiency had the most negative effect.
Biotin effectiveness decreases with decreasing nitrogen levels.
Vitamin deficiency negatively impacted the fermentation rates.
Minerals did not have a major impact.
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Biotin Deficiency and Nitrogen Levels
Excess NH4+ can be detrimental at no/low biotin concentrations
Summary of Effect of Combinatorial Nutritional Imbalance on Fermentations
N = Normal, S = Sluggish
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Metabolomic Comparison of Normal and Problematic Fermentation
Yeast cells are harvested from normal and problematic fermentations
Metabolites are extracted and
analyzed using GC‐MS‐TOF
The metabolite levels are
compared using MetaboAnalyst
software
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
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Mannitol
Sugar alcohol produced by plants and microbes.
Functions Carbohydrate storage depot Osmoprotectant Scavenger of reactive oxygen species (ROS)
Effect of Oxidative Stress on Fermentation
H2O2H2O2 H2O2H2O2
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Intracellular Mannitol Accumulation
Conclusions from Juice Composition and Metabolomic Study
Low YAN levels is not the cause of the problematic fermentation
Oxidative stress results in mannitol accumulation in yeast
High Pro:Arg ratio in the juice and mannitol accumulation in the
yeast from these juices are signals suggesting stress in the vines
Based on our study, nitrogen application was increased in the
vineyard for the next few years.
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Fermentation curves in DTF juice from consecutive vintages from the same vineyard after nitrogen application in the vineyard
Year 1
Year 2
Year 3
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+] ( has lower metabolic activity)
Study the effect of acetic acid and lactic acid produced by these
bacteria on yeast metabolic state
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Bacteria from Problematic Fermentations Acetic acid and Lactic acid bacteria isolated classified into 3 groups:
Included strains of Acetobacter pasteurianus, Gluconobacter cerinus, Gluconobacter oxydans, Lactobacillus kunkeii, Lactobacillus plantaram, Lactobacillus mali, Pediococcus damnosus.
• Inducing
• Inhibitory
• No effect
Both acetate and lactate levels produced vary across different genera
and within the same species.
A. No Effect B. Inhibiting C. InducingYeast spots
Bacterial spots
Figure 1
Result from Spent Media Assay
UCD 175 (Acetobacter pasteurianus) shows [GAR+] inducing
phenotype in spent media assay.
1) Spent GGM media
2) GGM media
Spent media induction assay Grow bacteria in media Sterile filter Inoculate yeast in spent media Screen on GGM plate
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Induction of [GAR+] prion state by acetic acidGGM plate
After Exposure to
No acetic acid
2% acetic acid
4% acetic acid
Different levels of acetic acid exposure elicit different metabolic response by yeast
Conclusions
Nitrogen supplementation using DAP should be done carefully taking into
account the other potential limiting nutrients in the must.
Oxidative stress results in mannitol accumulation in yeast. Chronic juice
issues may reflect stress in the vineyard
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.