ramakrishnan difficult to ferment juices
TRANSCRIPT
7/14/2016
1
Difficult to Ferment Juices
Vidhya Ramakrishnan
July 15, 2016
July 15, 2016
Factors impacting fermentations
7/14/2016
2
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
7/14/2016
3
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)
7/14/2016
4
Effect of Oxidative Stress on Fermentation
H2O2H2O2 H2O2H2O2
Intracellular Mannitol Accumulation
7/14/2016
5
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
7/14/2016
6
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
7/14/2016
7
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
7/14/2016
8
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
7/14/2016
9
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
7/14/2016
10
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
7/14/2016
11
Hydroxyproline (Tryptophan)C
on
cen
trat
ion
(m
g/L
)
Day0 Day1 Day2 Day3 Day4 Day6 Day10
Glycine (GABA)
7/14/2016
12
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!
7/14/2016
13
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